Antibiotics pp 291-306 | Cite as

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research

  • Sina Schäkermann
  • Pascal Prochnow
  • Julia E. Bandow
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1520)

Abstract

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

Key words

Mass spectrometry Ribosome heterogeneity Stress Proteomics 

Notes

Acknowledgments

We thank Birgit Klinkert and Johanna Roßmanith for the practical introduction into ribosome isolation and for technical support. Jennifer Stepanek and Dominik Wüllner are acknowledged for critically reading the manuscript. Furthermore, we would like to thank Dörte Becher and Knut Büttner for sharing mass spectrometry protocols. Funding from the German Federal State of North Rhine Westphalia (NRW) is acknowledged for the mass spectrometer (“Forschungsgroßgeräte der Länder”) used in this protocol. JEB acknowledges funding from NRW for the grant “Translation of innovative antibiotics from NRW.”

References

  1. 1.
    McCoy LS, Xie Y, Tor Y (2011) Antibiotics that target protein synthesis. Wiley Interdiscip Rev RNA 2(2):209–232CrossRefPubMedGoogle Scholar
  2. 2.
    Wilson DN (2014) Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat Rev Microbiol 12(1):35–48CrossRefPubMedGoogle Scholar
  3. 3.
    Byrgazov K, Vesper O, Moll I (2013) Ribosome heterogeneity: another level of complexity in bacterial translation regulation. Curr Opin Microbiol 16(2):133–139CrossRefPubMedPubMedCentralGoogle Scholar
  4. 5.
    Sauert M, Temmel H, Moll I (2015) Heterogeneity of the translational machinery: variations on a common theme. Biochimie 114:39–47CrossRefPubMedGoogle Scholar
  5. 6.
    Deusser E, Wittmann HG (1972) Ribosomal proteins: variation of the protein composition in Escherichia coli ribosomes as function of growth rate. Nature 238(5362):269–270CrossRefPubMedGoogle Scholar
  6. 8.
    Kurland CG, Voynow P, Hardy SJ, Randall L, Lutter L (1969) Physical and functional heterogeneity of E. coli ribosomes. Cold Spring Harb Symp Quant Biol 34:17–24CrossRefPubMedGoogle Scholar
  7. 9.
    Nanamiya H, Akanuma G, Natori Y, Murayama R, Kosono S, Kudo T, Kobayashi K, Ogasawara N, Park SM, Ochi K, Kawamura F (2004) Zinc is a key factor in controlling alternation of two types of L31 protein in the Bacillus subtilis ribosome. Mol Microbiol 52(1):273–283CrossRefPubMedGoogle Scholar
  8. 10.
    Natori Y, Nanamiya H, Akanuma G, Kosono S, Kudo T, Ochi K, Kawamura F (2007) A fail-safe system for the ribosome under zinc-limiting conditions in Bacillus subtilis. Mol Microbiol 63(1):294–307CrossRefPubMedGoogle Scholar
  9. 11.
    Agafonov DE, Kolb VA, Spirin AS (2001) Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage. EMBO Rep 2(5):399–402CrossRefPubMedPubMedCentralGoogle Scholar
  10. 12.
    Giangrossi M, Brandi A, Giuliodori AM, Gualerzi CO, Pon CL (2007) Cold-shock-induced de novo transcription and translation of infA and role of IF1 during cold adaptation. Mol Microbiol 64(3):807–821CrossRefPubMedGoogle Scholar
  11. 13.
    Giuliodori AM, Brandi A, Giangrossi M, Gualerzi CO, Pon CL (2007) Cold-stress-induced de novo expression of infC and role of IF3 in cold-shock translational bias. RNA 13(8):1355–1365CrossRefPubMedPubMedCentralGoogle Scholar
  12. 14.
    Wada A, Yamazaki Y, Fujita N, Ishihama A (1990) Structure and probable genetic location of a “ribosome modulation factor” associated with 100S ribosomes in stationary-phase Escherichia coli cells. Proc Natl Acad Sci U S A 87(7):2657–2661CrossRefPubMedPubMedCentralGoogle Scholar
  13. 16.
    Tagami K, Nanamiya H, Kazo Y, Maehashi M, Suzuki S, Namba E, Hoshiya M, Hanai R, Tozawa Y, Morimoto T, Ogasawara N, Kageyama Y, Ara K, Ozaki K, Yoshida M, Kuroiwa H, Kuroiwa T, Ohashi Y, Kawamura F (2012) Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp(0) mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome. Microbiologyopen 1(2):115–134CrossRefPubMedPubMedCentralGoogle Scholar
  14. 17.
    McKay SL, Portnoy DA (2015) Ribosome hibernation facilitates tolerance of stationary-phase bacteria to aminoglycosides. Antimicrob Agents Chemother 59(11):6992–6999CrossRefPubMedPubMedCentralGoogle Scholar
  15. 18.
    Kaberdina AC, Szaflarski W, Nierhaus KH, Moll I (2009) An unexpected type of ribosomes induced by kasugamycin: a look into ancestral times of protein synthesis? Mol Cell 33(2):227–236CrossRefPubMedPubMedCentralGoogle Scholar
  16. 19.
    Delvillani F, Papiani G, Deho G, Briani F (2011) S1 ribosomal protein and the interplay between translation and mRNA decay. Nucleic Acids Res 39(17):7702–7715CrossRefPubMedPubMedCentralGoogle Scholar
  17. 20.
    Vesper O, Amitai S, Belitsky M, Byrgazov K, Kaberdina AC, Engelberg-Kulka H, Moll I (2011) Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli. Cell 147(1):147–157CrossRefPubMedPubMedCentralGoogle Scholar
  18. 21.
    Mauro VP, Edelman GM (2002) The ribosome filter hypothesis. Proc Natl Acad Sci U S A 99(19):12031–12036CrossRefPubMedPubMedCentralGoogle Scholar
  19. 22.
    Akanuma G, Nanamiya H, Natori Y, Yano K, Suzuki S, Omata S, Ishizuka M, Sekine Y, Kawamura F (2012) Inactivation of ribosomal protein genes in Bacillus subtilis reveals importance of each ribosomal protein for cell proliferation and cell differentiation. J Bacteriol 194(22):6282–6291CrossRefPubMedPubMedCentralGoogle Scholar
  20. 23.
    Spedding G (1990) Isolation and analysis of ribosomes from prokaryotes, eukaryotes, and organelles. In: Spedding G (ed) Ribosomes and protein synthesis. Practical approach series. IRL Press, Oxford, UK, pp 1–29Google Scholar
  21. 24.
    Blaha G, Stelzl U, Spahn CM, Agrawal RK, Frank J, Nierhaus KH (2000) Preparation of functional ribosomal complexes and effect of buffer conditions on tRNA positions observed by cryoelectron microscopy. Methods Enzymol 317:292–309CrossRefPubMedGoogle Scholar
  22. 25.
    Silva JC, Gorenstein MV, Li GZ, Vissers JP, Geromanos SJ (2006) Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Mol Cell Proteomics 5(1):144–156CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Sina Schäkermann
    • 1
  • Pascal Prochnow
    • 1
  • Julia E. Bandow
    • 1
  1. 1.Applied MicrobiologyRuhr-Universität BochumBochumGermany

Personalised recommendations