Skip to main content
SpringerLink
Log in

The Activity of rRNA Resistance Methyltransferases Assessed by MALDI Mass Spectrometry

  • Protocol
New Antibiotic Targets

Part of the book series: Methods In Molecular Medicine™ ((MIMM,volume 142))

Summary

Resistance to antibiotics that target the bacterial ribosome is often conferred by methylation at specific nucleotides in the rRNA. The nucleotides that become methylated are invariably key sites of antibiotic interaction. The addition of methyl groups to each of these nucleotides is catalyzed by a specific methyltransferase enzyme. The Erm methyltransferases are a clinically prevalent group of enzymes that confer resistance to the therapeutically important macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. The target for Erm methyltransferases is at nucleotide A2058 in 23S rRNA, and methylation occurs before the rRNA has been assembled into 50S ribosomal particles. Erm methyltransferases occur in a phylogenetically wide range of bacteria and differ in whether they add one or two methyl groups to the A2058 target. The dimethylated rRNA confers a more extensive MLSB resistance phenotype. We describe here a method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the location and number of methyl groups added at any site in the rRNA. The method is particularly suited to studying in vitro methylation of RNA transcripts by resistance methyltransferases such as Erm.

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 139.00
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

Reference

  1. Gale, E. F., Cundliffe, E., Reynolds, P. E., Richmond, M. H., and Waring, M. J. (1981) The Molecular Basis of Antibiotic Action. John Wiley and Sons, London.

    Google Scholar 

  2. Vázquez, D. (1979) Inhibitors of Protein Biosynthesis. Springer-Verlag, Berlin.

    Google Scholar 

  3. Poehlsgaard, J., and Douthwaite, (2005) The bacterial ribosome as a target for antibiotics. Nat. Rev. Microbiol. 3, 870–881.

    Article  CAS  PubMed  Google Scholar 

  4. Cundliffe, E. (1990) Recognition sites for antibiotics within rRNA. InThe Ribosome: Structure, Function and Evolution (W. E. Hill, A. Dahlberg, .R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R.Warner, eds.). American Society for Microbiology, Washington DC, pp.479–490,

    Google Scholar 

  5. Green, R., and Noller, H. F. (1997) Ribosomes and translation. Ann. Rev. Biochem. 66, 679–716.

    Article  CAS  PubMed  Google Scholar 

  6. Nissen, P., Hansen, j., Ban, N., Moore, P. B.and Steitz, T. A. (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930.

    Article  CAS  PubMed  Google Scholar 

  7. Gregory, S. T., and Dahlberg, A. E (1999) Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23S ribosomal RNA. J. Mol. Biol. 289, 827–834.

    Article  CAS  PubMed  Google Scholar 

  8. Gabashvili, I. S., Gregory, S. T., Valle, M., Grassucci, R., Worbs, M., Wahl,M.C., Dahlberg, A. E., and Frank, J. (2001) The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22. Mol. Cell 8, 181–188.

    Article  CAS  PubMed  Google Scholar 

  9. Vester, B., and Douthwaite, S. (2001) Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob. Agents Chemother. 45, 1–12.

    Article  CAS  PubMed  Google Scholar 

  10. Douthwaite, S., Fourmy, D., and Yoshizawa, S. (2005) Nucleotide methylations in rRNA that confer resistance to ribosome-targeting antibiotics. In Fine-Tuning of RNA Functions by Modification and Editing (H. Grosjean, ed.), Vol.12. Springer, New York, pp.287–309.

    Google Scholar 

  11. Kagan, R. M., and Clarke, S. (1994) Widespread occurrence of three motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch. Biochem. Biophys. 310, 417–427.

    Article  CAS  PubMed  Google Scholar 

  12. Schubert, H. L., Blumenthal, R. M., and Cheng, X. (2003) Many paths to methyltransfer: A chronicle of convergence. Trends Biochem. Sci. 28, 329–335.

    Article  CAS  PubMed  Google Scholar 

  13. Skeggs, P. A., Thompson, J., and Cundliffe, E. (1985) Methylation of 16S ribosomal RNA and resistance to aminoglycoside antibiotics in clones of Streptomyces lividans carrying DNA from Streptomyces tenjimariensis. Mol. Gen. Genet. 200, 415–421.

    Article  CAS  PubMed  Google Scholar 

  14. Thompson, J., Skeggs, P. A., and Cundliffe, E. (1985) Methylation of 16S ribosomal RNA and resistance to the aminoglycoside antibiotics gentamicin and kanamycin determined by DNA from the gentamicin-producer, Micromonospora purpurea. Mol. Gen. Genet. 201, 168–173.

    Article  CAS  PubMed  Google Scholar 

  15. Wimberly, B. T., Brodersen, D. E., Clemons, W. M. J., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Structure of the 30S ribosomal subunit. Nature 407, 327–339.

    Article  CAS  PubMed  Google Scholar 

  16. Schl¨nzen, F., Tocilj, A., Zarivach, R., Harms, J., Gluehmann, M., Janell, D., Bashan, A., Bartels, H., Agmon, I., Franceschi, F., and Yonath, A. (2000) Structure of functionally activated small ribosomal subunit at 3.3 Angstroms resolution. Cell 102, 615–623.

    Article  Google Scholar 

  17. Liu, M., Kirpekar, F., van Wezel, G. P., and Douthwaite, S. (2000) The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA. Mol. Microbiol. 37, 811–820.

    Article  CAS  PubMed  Google Scholar 

  18. Thompson, J., Schmidt, F., and Cundliffe, E. (1982) Site of action of a ribosomal RNA methylase conferring resistance to thiostrepton. J. Biol. Chem. 257, 7915–7917.

    CAS  PubMed  Google Scholar 

  19. Bechthold, A., and Floss, H. G. (1994) Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2’-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA. Eur. J. Biochem. 224, 431–437.

    Article  CAS  PubMed  Google Scholar 

  20. Skinner, R., Cundliffe, E., and Schmidt, F. J. (1983) Site of action of a ribosomal RNA methylase responsible for resistance to erythromycin and other antibiotics. J. Biol. Chem. 258, 12702–12706.

    CAS  PubMed  Google Scholar 

  21. Vester, B., and Douthwaite, S. (1994) Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase. J. Bacteriol. 176, 6999–7004.

    CAS  PubMed  Google Scholar 

  22. Mann, P. A., Xiong, L., Mankin, A. S., Chau, A. S., Mendrick, C. A., Najarian,D.J., Cramer, C. A., Loebenberg, D., Coates, E., Murgolo, N. J., Aarestrup, F. M., Goering, R. V., Black, T. A., Hare, R. S., and McNicholas,P.M. (2001) EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance. Mol. Microbiol. 41, 1349–1356.

    Article  CAS  PubMed  Google Scholar 

  23. Treede, I., Jakobsen, L., Kirpekar, F., Vester, B., Weitnauer, G. A. B., and Douthwaite, S. (2003) The avilamycin resistance determinants AviRa and AviRb methylate 23S rRNA at the guanosine 2535 base and the uridine 2479 ribose. Mol. Microbiol. 49, 309–318.

    Article  CAS  PubMed  Google Scholar 

  24. Weisblum, B. (1995) Erythromycin resistance by ribosome modification. Antimicrob. Agents Chemother. 39, 577–585.

    CAS  PubMed  Google Scholar 

  25. Zalacain, M., and Cundliffe, E. (1990) Methylation of 23S ribosomal RNA due to carB, an antibiotic-resistance determinant from the carbomycin producer, Streptomyces thermotolerans. Eur. J. Biochem. 189, 67–72.

    Article  CAS  PubMed  Google Scholar 

  26. Liu, M., and Douthwaite, S. (2002) Activity of the ketolide antibiotic telithromycin is refractory to Erm monomethylation of bacterial rRNA. Antimicrob. Agents Chemother. 46, 1629–1633.

    Article  CAS  PubMed  Google Scholar 

  27. Roberts, M. C., Sutcliffe, J., Courvalin, P., Jensen, L. B., Rood, J., and Sepp"alä, H. (1999) Nomenclature for macrolide and macrolide-lincomycin-streptogramin B resistance determinants. Antimicrob. Agents Chemother. 43, 2823–2830.

    CAS  PubMed  Google Scholar 

  28. Champney, W. S., Chittum, H. S., and Tober, C. L. (2003) A 50S ribosomal subunit precursor particle is a substrate for the ErmC methyltransferase in Staphylococcus aureus cells. Curr. Microbiol. 46, 453–460.

    Article  CAS  PubMed  Google Scholar 

  29. Vester, B., Nielsen, A. K., Hansen, L. H., and Douthwaite, S. (1998) ErmE methyltransferase recognition elements in RNA substrates. J. Mol. Biol. 282, 255–264.

    Article  CAS  PubMed  Google Scholar 

  30. Kovalic, D., Giannattasio, R. B., Jin, H. J., and Weisblum, B. (1994) 23S rRNA domain V, a fragment that can be specifically methylated in vitro by the ErmSF (TlrA) methyltransferase. J. Bacteriol. 176, 6992–6998.

    CAS  PubMed  Google Scholar 

  31. Kirpekar, F., Douthwaite, S., and Roepstorff, P. (2000) Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry. RNA 6, 296–306.

    Article  CAS  PubMed  Google Scholar 

  32. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989).it Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, New York.

    Google Scholar 

  33. Vester, B., Hansen, L. H., and Douthwaite, S. (1995) The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase. RNA 1, 501–509.

    CAS  PubMed  Google Scholar 

  34. Vilsen, I. D., Vester, B., and Douthwaite, S. (1999) ErmE methyltransferase rocognizes features of the primary and secondary structure in a motif within domain V of 23S rRNA. J. Mol. Biol. 286, 365–374.

    Article  Google Scholar 

  35. Denoya, C., and Dubnau, D. (1989) Mono- and dimethylating activities and kinetic studies of the ermC 23 S rRNA methyltransferase. J. Biol. Chem. 264, 2615–2624.

    CAS  PubMed  Google Scholar 

  36. Douthwaite, S., Jalava, J., and Jakobsen, L. (2005) Ketolide resistance in Streptococcus pyogenes correlates with the degree of rRNA dimethylation by Erm. Mol. Microbiol. 58, 613–622.

    Article  CAS  PubMed  Google Scholar 

  37. Madsen, C. T., Jakobsen, L., and Douthwaite, S. (2005) Mycobacterium smegmatis Erm(38) is a reluctant dimethyltransferase. Antimicrob. Agents Chemother. 49, 3803–3809.

    Article  CAS  PubMed  Google Scholar 

  38. Mengel-Jorgensen, J., Jensen, S. S., Rasmussen, A., Poehlsgaard, J., Iversen,J.J., and Kirpekar, F. (2006) Modifications in Thermus thermophilus 23S ribosomal RNA are centered in regions of RNA-RNA contact. J. Biol. Chem. 281, 22108–22117.

    Article  CAS  PubMed  Google Scholar 

  39. Asara, J. M., and Allison, J. (1999) Enhanced detection of oligonucleotides in UV MALDI MS using the tetraamine spermine as a matrix additive. Anal. Chem. 71, 2866–2870.

    Article  CAS  PubMed  Google Scholar 

  40. Zhu, Y. F., Chung, C. N., Taranenko, N. I., Allman, S. L., Martin, S. A., Haff, L., and Chen, C. H. (1996) The study of 2,3,4-trihydroxyacetophenone and 2,4,6-trihydroxyacetophenone as matrices for DNA detection in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 10, 383–388.

    Article  CAS  Google Scholar 

  41. Kirpekar, F., and Krogh, T. N. (2001) RNA fragmentation studied in a matrix-assisted laser desorption/ionisation tandem quadrupole/orthogonal time-of-flight mass spectrometer. Rapid Comm. Mass Spectrom. 15, 8–14.

    Article  CAS  Google Scholar 

  42. McLuckey, S. A., Van Berkel, G. J., and Glish, G. L. (1992) Tandem mass spectrometry of small multiply charged oligonucleotides. J. Amer. Soc. Mass Spectrom. 3, 60–70.

    Article  CAS  Google Scholar 

  43. Cannone, J. J., Subramanian, S., Schnare, M. N., Collett, J. R., D’Souza, L. M., Du, Y., Feng, B., Lin, N., Madabusi, L. V., M"uller, K. M., Pande, N., Shang, Z., Yu, N., and Gutell, R. R. (2002) The Comparative RNA Web (CRW) Site: An online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3, 2.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Birte Vester and Lykke Haastrup Hansen for discussions. Support from the Danish Research Agency (FNU Grants #21-04-0505 and #21-04-0520), the Nucleic Acid Center of the Danish Grundforskningsfond, and CDC funds are gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej, Denmark

    Stephen Douthwaite, Rikke Lind Jensen & Finn Kirpekar

Authors
  1. Stephen Douthwaite
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rikke Lind Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Finn Kirpekar
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, East Tennessee State University, Johnson, Tennessee

    W. Scott Champney

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press Inc.

About this protocol

Cite this protocol

Douthwaite, S., Jensen, R.L., Kirpekar, F. (2008). The Activity of rRNA Resistance Methyltransferases Assessed by MALDI Mass Spectrometry. In: Champney, W.S. (eds) New Antibiotic Targets. Methods In Molecular Medicine™, vol 142. Humana Press. https://doi.org/10.1007/978-1-59745-246-5_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-246-5_18

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-915-4

  • Online ISBN: 978-1-59745-246-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation