Abstract
Bioinformatic approaches and a large volume of prokaryotic genome sequences have enabled rapid identification of regulatory proteins with features to bind DNA or RNA in a given prokaryote. However, biological relevance of these regulatory proteins requires methods to rapidly purify and determine their binding properties within the physiological context or life style of the organism. Here, we describe the experimental approaches to determine the nucleic acid binding properties of regulatory proteins of Borrelia burgdorferi using Borrelia host-adaptation Re.3gulator (BadR—a DNA binding protein) and Carbon storage regulators A of B. b urgdorferi (CsrABb—an RNA binding protein) as examples. Best laboratory practices associated with overexpression/purification of recombinant borrelial proteins, synthesis of target nucleic acid sequences, and electrophoretic mobility assays to assess the protein/nucleic acid interactions are described. The methods described are intended to facilitate empirical assessment of the binding affinity, co-factor requirements, quality of the interacting partners, and readily modifiable assay conditions to assess the binding properties to define known and unknown regulatory properties of nucleic acid binding proteins of B. burgdorferi.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Samuels DS (2011) Gene regulation in Borrelia burgdorferi. Annu Rev Microbiol 65:479–499
Radolf JD, Caimano MJ, Stevenson B, Hu LT (2012) Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 10:87–99
Seshu J, Boylan JA, Hyde JA, Swingle KL, Gherardini FC, Skare JT (2004) A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi. Mol Microbiol 54:1352–1363
Miller CL, Karna SL, Seshu J (2013) Borrelia host adaptation regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi. Mol Microbiol 88:105–124
Karna SL, Prabhu RG, Lin YH, Miller CL, Seshu J (2013) Contributions of environmental signals and conserved residues to the functions of carbon storage regulator a of Borrelia burgdorferi. Infect Immun 81:2972–2985
Karna SL, Sanjuan E, Esteve-Gassent MD, Miller CL, Maruskova M, Seshu J (2011) CsrA modulates levels of lipoproteins and key regulators of gene expression critical for pathogenic mechanisms of Borrelia burgdorferi. Infect Immun 79:732–744
Esteve-Gassent MD, Elliott NL, Seshu J (2009) sodA is essential for virulence of Borrelia burgdorferi in the murine model of Lyme disease. Mol Microbiol 71:594–612
Esteve-Gassent MD, Smith TC 2nd, Small CM, Thomas DP, Seshu J (2015) Absence of sodA increases the levels of oxidation of key metabolic determinants of Borrelia burgdorferi. PLoS One 10:e0136707
Ouyang Z, Zhou J, Brautigam CA, Deka R, Norgard MV (2014) Identification of a core sequence for the binding of BosR to the rpoS promoter region in Borrelia burgdorferi. Microbiology 160:851–862
Boylan JA, Posey JE, Gherardini FC (2003) Borrelia oxidative stress response regulator, BosR: a distinctive Zn-dependent transcriptional activator. Proc Natl Acad Sci U S A 100:11684–11689
Maruskova M, Esteve-Gassent MD, Sexton VL, Seshu J (2008) Role of the BBA64 locus of Borrelia burgdorferi in early stages of infectivity in a murine model of Lyme disease. Infect Immun 76:391–402
Maruskova M, Seshu J (2008) Deletion of BBA64, BBA65, and BBA66 loci does not alter the infectivity of Borrelia burgdorferi in the murine model of Lyme disease. Infect Immun 76:5274–5284
Ouyang Z, Zhou J (2015) BadR (BB0693) controls growth phase-dependent induction of rpoS and bosR in Borrelia burgdorferi via recognizing TAAAATAT motifs. Mol Microbiol 98:1147–1167. doi:10.1111/mmi.13206
Labandeira-Rey M, Seshu J, Skare JT (2003) The absence of linear plasmid 25 or 28-1 of Borrelia burgdorferi dramatically alters the kinetics of experimental infection via distinct mechanisms. Infect Immun 71:4608–4613
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
Hyde JA, Weening EH, Skare JT (2011) Genetic transformation of Borrelia burgdorferi. Curr Protoc Microbiol Chapter 12:Unit 12C 14
Van Laar TA, Lin YH, Miller CL, Karna SL, Chambers JP, Seshu J (2012) Effect of levels of acetate on the mevalonate pathway of Borrelia burgdorferi. PLoS One 7:e38171
Acknowledgments
This study was partly supported by Public Health Service grant SC1-AI-078559 from the National Institute of Allergy and Infectious Diseases, the Army Research Office of the Department of Defense under Contract No. W911NF-11-1-0136, pre-doctoral fellowships from the South Texas Center for Emerging Infectious Diseases (YHL, TAV, SLRK), and the Center For Excellence in Infection Genomics (TCS, CLM, TAV). TCS is supported by a pre-doctoral fellowship from The Brown Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Seshu, J., Smith, T.C., Lin, YH., Karna, S.L.R., Miller, C.L., Van Laar, T. (2018). Analysis of DNA and RNA Binding Properties of Borrelia burgdorferi Regulatory Proteins. 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_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7383-5_13
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