Identification of Acetylated Proteins in Borrelia burgdorferi

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


Posttranslational modification (PTM) of proteins has emerged as a major regulatory mechanism in all three domains of life. One emerging PTM is Nε-lysine acetylation—the acetylation of the epsilon amino group of lysine residues. Nε-lysine acetylation is known to regulate multiple cellular processes. In eukaryotes, it regulates chromatin structure, transcription, metabolism, signal transduction, and the cytoskeleton. Recently, multiple groups have detected Nε-lysine acetylation in diverse bacterial phyla, but no work on protein acetylation in Borrelia burgdorferi has been reported. Here, we describe a step-by-step protocol to identify Nε-lysine acetylated proteins in B. burgdorferi.

Key words

Borrelia burgdorferi Posttranslational modification Protein acetylation Immunoprecipitation Acetylated lysine protein 



This work was supported by grants from the HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI4684064 to XFY).


  1. 1.
    Glozak MA, Seto E (2007) Histone deacetylases and cancer. Oncogene 26:5420–5432CrossRefPubMedGoogle Scholar
  2. 2.
    Rardin MJ, Newman JC, Held JM, Cusack MP, Sorensen DJ, Li B, Schilling B, Mooney SD, Kahn CR, Verdin E, Gibson BW (2013) Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways. Proc Natl Acad Sci U S A 110:6601–6606CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Yang XJ, Seto E (2008) Lysine acetylation: codified crosstalk with other posttranslational modifications. Mol Cell 31:449–461CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Verdin E, Ott M (2015) 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond. Nat Rev Mol Cell Biol 16:258–264CrossRefPubMedGoogle Scholar
  5. 5.
    Kim D, Yu BJ, Kim JA, Lee YJ, Choi SG, Kang S, Pan JG (2013) The acetylproteome of gram-positive model bacterium Bacillus subtilis. Proteomics 13:1726–1736CrossRefPubMedGoogle Scholar
  6. 6.
    Lee DW, Kim D, Lee YJ, Kim JA, Choi JY, Kang S, Pan JG (2013) Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus. Proteomics 13:2278–2282CrossRefPubMedGoogle Scholar
  7. 7.
    Wang Q, Zhang Y, Yang C, Xiong H, Lin Y, Yao J, Li H, Xie L, Zhao W, Yao Y, Ning ZB, Zeng R, Xiong Y, Guan KL, Zhao S, Zhao GP (2010) Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science 327:1004–1007CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Weinert BT, Iesmantavicius V, Wagner SA, Scholz C, Gummesson B, Beli P, Nystrom T, Choudhary C (2013) Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli. Mol Cell 51:265–272CrossRefPubMedGoogle Scholar
  9. 9.
    Wu X, Vellaichamy A, Wang D, Zamdborg L, Kelleher NL, Huber SC, Zhao Y (2013) Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics. J Proteome 79:60–71CrossRefGoogle Scholar
  10. 10.
    Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y (2009) Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli. Mol Cell Proteomics 8:215–225CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhang K, Zheng S, Yang JS, Chen Y, Cheng Z (2013) Comprehensive profiling of protein lysine acetylation in Escherichia coli. J Proteome Res 12:844–851CrossRefPubMedGoogle Scholar
  12. 12.
    Kuhn ML, Zemaitaitis B, Hu LI, Sahu A, Sorensen D, Minasov G, Lima BP, Scholle M, Mrksich M, Anderson WF, Gibson BW, Schilling B, Wolfe AJ (2014) Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation. PLoS One 9:e94816. doi: 10.1371/journal.pone.0094816. eCollection 2014CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Yu B, Kim J, Moon J, Ryu S, Pan J (2008) The diversity of lysine-acetylated proteins in Escherichia coli. J Microbiol Biotechnol 18:1529–1536PubMedGoogle Scholar
  14. 14.
    Baeza J, Dowell JA, Smallegan MJ, Fan J, Amador-Noguez D, Khan Z, Denu JM (2014) Stoichiometry of site-specific lysine acetylation in an entire proteome. J Biol Chem 289:21326–21338CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kosono S, Tamura M, Suzuki S, Kawamura Y, Yoshida A, Nishiyama M, Yoshida M (2015) Changes in the acetylome and Succinylome of Bacillus subtilis in response to carbon source. PLoS One 10:e0131169. doi: 10.1371/journal.pone.0131169. eCollection 2015CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hu LI, Lima BP, Wolfe AJ (2010) Bacterial protein acetylation: the dawning of a new age. Mol Microbiol 77:15–21CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Jones JD, O'Connor CD (2011) Protein acetylation in prokaryotes. Proteomics 11:3012–3022CrossRefPubMedGoogle Scholar
  18. 18.
    Kim GW, Yang XJ (2011) Comprehensive lysine acetylomes emerging from bacteria to humans. Trends Biochem Sci 36:211–220CrossRefPubMedGoogle Scholar
  19. 19.
    Soppa J (2010) Protein acetylation in archaea, bacteria, and eukaryotes. Archaea. doi: 10.1155/2010/820681
  20. 20.
    Thao S, Escalante-Semerena JC (2011) Control of protein function by reversible N[epsilon]-lysine acetylation in bacteria. Curr Opin Microbiol 14:200–204CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hentchel KL, Escalante-Semerena JC (2015) Acylation of biomolecules in prokaryotes: a widespread strategy for the control of biological function and metabolic stress. Microbiol Mol Biol Rev 79:321–346CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Bernal V, Castano-Cerezo S, Gallego-Jara J, Ecija-Conesa A, de Diego T, Iborra JL, Canovas M (2014) Regulation of bacterial physiology by lysine acetylation of proteins. New Biotechnol 31:586–595CrossRefGoogle Scholar
  23. 23.
    Blander G, Guarente L (2004) The SIR2 family of protein deacetylases. Annu Rev Biochem 73:417–435CrossRefPubMedGoogle Scholar
  24. 24.
    Hentchel KL, Thao S, Intile PJ, Escalante-Semerena JC (2015) Deciphering the regulatory circuitry that controls reversible lysine acetylation in salmonella enterica. MBio 6:e00891. doi: 10.1128/mBio.00891-15 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Yang XJ, Seto E (2008) The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell Biol 9:206–218CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Barbour AG (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med 57:521–525PubMedPubMedCentralGoogle Scholar
  27. 27.
    Ngoka LC (2008) Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers. Proteome Sci 6:30CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Yang Y, Liu Y, Dong Z, Xu J, Peng H, Liu Z, Zhang JT (2007) Regulation of function by dimerization through the amino-terminal membrane-spanning domain of human ABCC1/MRP1. J Biol Chem 282:8821–8830CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Microbiology and ImmunologyIndiana University School of MedicineIndianapolisUSA
  2. 2.Department of Microbiology and ImmunologyLoyola University Chicago, Stritch School of MedicineMaywoodUSA

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