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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Reference
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.
VƔzquez, D. (1979) Inhibitors of Protein Biosynthesis. Springer-Verlag, Berlin.
Poehlsgaard, J., and Douthwaite, (2005) The bacterial ribosome as a target for antibiotics. Nat. Rev. Microbiol. 3, 870ā881.
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.479ā490,
Green, R., and Noller, H. F. (1997) Ribosomes and translation. Ann. Rev. Biochem. 66, 679ā716.
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, 920ā930.
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, 827ā834.
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, 181ā188.
Vester, B., and Douthwaite, S. (2001) Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob. Agents Chemother. 45, 1ā12.
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.287ā309.
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, 417ā427.
Schubert, H. L., Blumenthal, R. M., and Cheng, X. (2003) Many paths to methyltransfer: A chronicle of convergence. Trends Biochem. Sci. 28, 329ā335.
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, 415ā421.
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, 168ā173.
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, 327ā339.
SchlĀØnzen, 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, 615ā623.
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, 811ā820.
Thompson, J., Schmidt, F., and Cundliffe, E. (1982) Site of action of a ribosomal RNA methylase conferring resistance to thiostrepton. J. Biol. Chem. 257, 7915ā7917.
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 2ā-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA. Eur. J. Biochem. 224, 431ā437.
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, 12702ā12706.
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, 6999ā7004.
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, 1349ā1356.
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, 309ā318.
Weisblum, B. (1995) Erythromycin resistance by ribosome modification. Antimicrob. Agents Chemother. 39, 577ā585.
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, 67ā72.
Liu, M., and Douthwaite, S. (2002) Activity of the ketolide antibiotic telithromycin is refractory to Erm monomethylation of bacterial rRNA. Antimicrob. Agents Chemother. 46, 1629ā1633.
Roberts, M. C., Sutcliffe, J., Courvalin, P., Jensen, L. B., Rood, J., and Sepp"alƤ, H. (1999) Nomenclature for macrolide and macrolide-lincomycin-streptogramin B resistance determinants. Antimicrob. Agents Chemother. 43, 2823ā2830.
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, 453ā460.
Vester, B., Nielsen, A. K., Hansen, L. H., and Douthwaite, S. (1998) ErmE methyltransferase recognition elements in RNA substrates. J. Mol. Biol. 282, 255ā264.
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, 6992ā6998.
Kirpekar, F., Douthwaite, S., and Roepstorff, P. (2000) Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry. RNA 6, 296ā306.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989).it Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, New York.
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, 501ā509.
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, 365ā374.
Denoya, C., and Dubnau, D. (1989) Mono- and dimethylating activities and kinetic studies of the ermC 23āS rRNA methyltransferase. J. Biol. Chem. 264, 2615ā2624.
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, 613ā622.
Madsen, C. T., Jakobsen, L., and Douthwaite, S. (2005) Mycobacterium smegmatis Erm(38) is a reluctant dimethyltransferase. Antimicrob. Agents Chemother. 49, 3803ā3809.
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, 22108ā22117.
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, 2866ā2870.
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, 383ā388.
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, 8ā14.
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, 60ā70.
Cannone, J. J., Subramanian, S., Schnare, M. N., Collett, J. R., DāSouza, 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.
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
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
Ā© 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 Medicineā¢, 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