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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
McCoy LS, Xie Y, Tor Y (2011) Antibiotics that target protein synthesis. Wiley Interdiscip Rev RNA 2(2):209–232
Wilson DN (2014) Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat Rev Microbiol 12(1):35–48
Byrgazov K, Vesper O, Moll I (2013) Ribosome heterogeneity: another level of complexity in bacterial translation regulation. Curr Opin Microbiol 16(2):133–139
Sauert M, Temmel H, Moll I (2015) Heterogeneity of the translational machinery: variations on a common theme. Biochimie 114:39–47
Deusser E, Wittmann HG (1972) Ribosomal proteins: variation of the protein composition in Escherichia coli ribosomes as function of growth rate. Nature 238(5362):269–270
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 34:17–24
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):273–283
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):294–307
Agafonov DE, Kolb VA, Spirin AS (2001) Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage. EMBO Rep 2(5):399–402
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):807–821
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):1355–1365
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):2657–2661
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):115–134
McKay SL, Portnoy DA (2015) Ribosome hibernation facilitates tolerance of stationary-phase bacteria to aminoglycosides. Antimicrob Agents Chemother 59(11):6992–6999
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):227–236
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):7702–7715
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):147–157
Mauro VP, Edelman GM (2002) The ribosome filter hypothesis. Proc Natl Acad Sci U S A 99(19):12031–12036
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):6282–6291
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 1–29
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 317:292–309
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):144–156
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.”
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Schäkermann, S., Prochnow, P., Bandow, J.E. (2017). Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research. In: Sass, P. (eds) Antibiotics. Methods in Molecular Biology, vol 1520. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6634-9_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6634-9_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6632-5
Online ISBN: 978-1-4939-6634-9
eBook Packages: Springer Protocols