1887

Abstract

N -lysine acetylation and succinylation are ubiquitous post-translational modifications in eukaryotes and bacteria. In the present study, we showed a dynamic change in acetylation and succinylation of TufA, the translation elongation factor Tu, from Bacillus subtilis. Increased acetylation of TufA was observed during the exponential growth phase in LB and minimal glucose conditions, and its acetylation level decreased upon entering the stationary phase, while its succinylation increased during the late stationary phase. TufA was also succinylated during vegetative growth under minimal citrate or succinate conditions. Mutational analysis showed that triple succinylation mimic mutations at Lys306, Lys308 and Lys316 in domain-3 of TufA had a negative effect on B. subtilis growth, whereas the non-acylation mimic mutations at these three lysine residues did not. Consistent with the growth phenotypes, the triple succinylation mimic mutant showed 67 % decreased translation activity in vitro, suggesting a possibility that succinylation at the lysine residues in domain-3 decreases the translation activity. TufA, including Lys308, was non-enzymatically succinylated by physiological concentrations of succinyl-CoA. Lys42 in the G-domain was identified as the most frequently modified acetylation site, though its acetylation was likely dispensable for TufA translation activity and growth. Determination of the intracellular levels of acetylating substrates and TufA acetylation revealed that acetyl phosphate was responsible for acetylation at several lysine sites of TufA, but not for Lys42 acetylation. It was speculated that acetyl-CoA was likely responsible for Lys42 acetylation, though AcuA acetyltransferase was not involved. Zn-dependent AcuC and NAD-dependent SrtN deacetylases were responsible for deacetylation of TufA, including Lys42. These findings suggest the potential regulatory roles of acetylation and succinylation in controlling TufA function and translation in response to nutrient environments in B. subtilis.

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2018-11-05
2020-02-25
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References

  1. Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 2014;15:536–550 [CrossRef][PubMed]
    [Google Scholar]
  2. Hentchel KL, Escalante-Semerena JC. Acylation of biomolecules in prokaryotes: a widespread strategy for the control of biological function and metabolic stress. Microbiol Mol Biol Rev 2015;79:321–346 [CrossRef][PubMed]
    [Google Scholar]
  3. Carabetta VJ, Cristea IM. Regulation, function, and detection of protein acetylation in bacteria. J Bacteriol 2017;199:e0010700117 [CrossRef][PubMed]
    [Google Scholar]
  4. Weinert BT, Schölz C, Wagner SA, Iesmantavicius V, Su D et al. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell Rep 2013;4:842–851 [CrossRef][PubMed]
    [Google Scholar]
  5. Wagner GR, Payne RM. Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix. J Biol Chem 2013;288:29036–29045 [CrossRef][PubMed]
    [Google Scholar]
  6. Okanishi H, Kim K, Fukui K, Yano T, Kuramitsu S et al. Proteome-wide identification of lysine succinylation in thermophilic and mesophilic bacteria. Biochim Biophys Acta Proteins Proteom 2017;1865:232–242 [CrossRef][PubMed]
    [Google Scholar]
  7. Weinert BT, Iesmantavicius V, Wagner SA, Schölz C, Gummesson B et al. Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli. Mol Cell 2013;51:265–272 [CrossRef][PubMed]
    [Google Scholar]
  8. Schilling B, Christensen D, Davis R, Sahu AK, Hu LI et al. Protein acetylation dynamics in response to carbon overflow in Escherichia coli. Mol Microbiol 2015;98:847–863 [CrossRef][PubMed]
    [Google Scholar]
  9. Kosono S, Tamura M, Suzuki S, Kawamura Y, Yoshida A et al. Changes in the acetylome and succinylome of Bacillus subtilis in response to carbon source. PLoS One 2015;10:e0131169 [CrossRef][PubMed]
    [Google Scholar]
  10. Yang M, Wang Y, Chen Y, Cheng Z, Gu J et al. Succinylome analysis reveals the involvement of lysine succinylation in metabolism in pathogenic Mycobacterium tuberculosis. Mol Cell Proteomics 2015;14:796–811 [CrossRef][PubMed]
    [Google Scholar]
  11. Mizuno Y, Nagano-Shoji M, Kubo S, Kawamura Y, Yoshida A et al. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction. Microbiologyopen 2016;5:152–173 [CrossRef][PubMed]
    [Google Scholar]
  12. Carabetta VJ, Greco TM, Tanner AW, Cristea IM, Dubnau D. Temporal regulation of the Bacillus subtilis acetylome and evidence for a role of mreb acetylation in cell wall growth. mSystems 2016;1:e0000500016 [CrossRef][PubMed]
    [Google Scholar]
  13. Ishigaki Y, Akanuma G, Yoshida M, Horinouchi S, Kosono S et al. Protein acetylation involved in streptomycin biosynthesis in Streptomyces griseus. J Proteomics 2017;155:63–72 [CrossRef][PubMed]
    [Google Scholar]
  14. Sadoul K, Wang J, Diagouraga B, Khochbin S. The tale of protein lysine acetylation in the cytoplasm. J Biomed Biotechnol 2011;2011:970382 [CrossRef][PubMed]
    [Google Scholar]
  15. Kuhn ML, Zemaitaitis B, Hu LI, Sahu A, Sorensen D et al. Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation. PLoS One 2014;9:e94816 [CrossRef][PubMed]
    [Google Scholar]
  16. Weinert BT, Satpathy S, Hansen BK, Lyon D, Jensen LJ et al. Accurate quantification of site-specific acetylation stoichiometry reveals the impact of sirtuin deacetylase CobB on the E. coli acetylome. Mol Cell Proteomics 2017;16:759–769 [CrossRef][PubMed]
    [Google Scholar]
  17. Yoshida M, Kudo N, Kosono S, Ito A. Chemical and structural biology of protein lysine deacetylases. Proc Jpn Acad Ser B Phys Biol Sci 2017;93:297–321 [CrossRef][PubMed]
    [Google Scholar]
  18. Tu S, Guo S-J, Chen C-S, Liu C-X, Jiang H-W et al. YcgC represents a new protein deacetylase family in prokaryotes. eLife 2016;4:e05322 [CrossRef]
    [Google Scholar]
  19. Kremer M, Kuhlmann N, Lechner M, Baldus L, Lammers M. Comment on 'YcgC represents a new protein deacetylase family in prokaryotes'. eLife 2018;7:e37798 [CrossRef][PubMed]
    [Google Scholar]
  20. Colak G, Xie Z, Zhu AY, Dai L, Lu Z et al. Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli. Mol Cell Proteomics 2013;12:3509–3520 [CrossRef][PubMed]
    [Google Scholar]
  21. Gardner JG, Grundy FJ, Henkin TM, Escalante-Semerena JC. Control of acetyl-coenzyme A synthetase (AcsA) activity by acetylation/deacetylation without NAD+ involvement in Bacillus subtilis. J Bacteriol 2006;188:5460–5468 [CrossRef][PubMed]
    [Google Scholar]
  22. Gardner JG, Escalante-Semerena JC. Biochemical and mutational analyses of AcuA, the acetyltransferase enzyme that controls the activity of the acetyl coenzyme a synthetase (AcsA) in Bacillus subtilis. J Bacteriol 2008;190:5132–5136 [CrossRef][PubMed]
    [Google Scholar]
  23. Gardner JG, Escalante-Semerena JC. In Bacillus subtilis, the sirtuin protein deacetylase, encoded by the srtN gene (formerly yhdZ), and functions encoded by the acuABC genes control the activity of acetyl coenzyme A synthetase. J Bacteriol 2009;191:1749–1755 [CrossRef][PubMed]
    [Google Scholar]
  24. Ogura M, Asai K. Glucose induces ECF sigma factor genes, sigX and sigM, independent of cognate anti-sigma factors through acetylation of CshA in Bacillus subtilis. Front Microbiol 2016;7:1918 [CrossRef][PubMed]
    [Google Scholar]
  25. Kim D, Yu BJ, Kim JA, Lee YJ, Choi SG et al. The acetylproteome of Gram-positive model bacterium Bacillus subtilis. Proteomics 2013;13:1726–1736 [CrossRef][PubMed]
    [Google Scholar]
  26. Ye Q, Ji QQ, Yan W, Yang F, Wang ED. Acetylation of lysine ϵ-amino groups regulates aminoacyl-tRNA synthetase activity in Escherichia coli. J Biol Chem 2017;292:10709–10722 [CrossRef][PubMed]
    [Google Scholar]
  27. Venkat S, Gregory C, Gan Q, Fan C. Biochemical characterization of the lysine acetylation of tyrosyl-trna synthetase in Escherichia coli. Chembiochem 2017;18:1928–1934 [CrossRef][PubMed]
    [Google Scholar]
  28. Cao X, Li C, Xiao S, Tang Y, Huang J et al. Acetylation promotes TyrRS nuclear translocation to prevent oxidative damage. Proc Natl Acad Sci USA 2017;114:687–692 [CrossRef][PubMed]
    [Google Scholar]
  29. Ishfaq M, Maeta K, Maeda S, Natsume T, Ito A et al. Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A). FEBS Lett 2012;586:3236–3241 [CrossRef][PubMed]
    [Google Scholar]
  30. Kosono S, Ohashi Y, Kawamura F, Kitada M, Kudo T. Function of a principal Na+/H+ antiporter, ShaA, is required for initiation of sporulation in Bacillus subtilis. J Bacteriol 2000;182:898–904 [CrossRef][PubMed]
    [Google Scholar]
  31. Akanuma G, Suzuki S, Yano K, Nanamiya H, Natori Y et al. Single mutations introduced in the essential ribosomal proteins L3 and S10 cause a sporulation defect in Bacillus subtilis. J Gen Appl Microbiol 2013;59:105–117 [CrossRef][PubMed]
    [Google Scholar]
  32. Sato T, Harada K, Kobayashi Y. Analysis of suppressor mutations of spoIVCA mutations: occurrence of DNA rearrangement in the absence of site-specific DNA recombinase spoIVCA in Bacillus subtilis. J Bacteriol 1996;178:3380–3383 [CrossRef][PubMed]
    [Google Scholar]
  33. Imamura D, Kobayashi K, Sekiguchi J, Ogasawara N, Takeuchi M et al. spoIVH (ykvV), a requisite cortex formation gene, is expressed in both sporulating compartments of Bacillus subtilis. J Bacteriol 2004;186:5450–5459 [CrossRef][PubMed]
    [Google Scholar]
  34. Natori Y, Nanamiya H, Akanuma G, Kosono S, Kudo T et al. A fail-safe system for the ribosome under zinc-limiting conditions in Bacillus subtilis. Mol Microbiol 2007;63:294–307 [CrossRef][PubMed]
    [Google Scholar]
  35. Prüss BM, Wolfe AJ. Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli. Mol Microbiol 1994;12:973–984[PubMed]
    [Google Scholar]
  36. Ramos-Montañez S, Kazmierczak KM, Hentchel KL, Winkler ME. Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39. J Bacteriol 2010;192:6390–6400 [CrossRef][PubMed]
    [Google Scholar]
  37. Jez JM, Ferrer JL, Bowman ME, Dixon RA, Noel JP. Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 2000;39:890–902 [CrossRef][PubMed]
    [Google Scholar]
  38. Gao H, Jiang X, Pogliano K, Aronson AI. The E1beta and E2 subunits of the Bacillus subtilis pyruvate dehydrogenase complex are involved in regulation of sporulation. J Bacteriol 2002;184:2780–2788 [CrossRef][PubMed]
    [Google Scholar]
  39. Hartman H, Smith TF. GTPases and the origin of the ribosome. Biol Direct 2010;5:36 [CrossRef][PubMed]
    [Google Scholar]
  40. Tubulekas I, Hughes D. Growth and translation elongation rate are sensitive to the concentration of EF-Tu. Mol Microbiol 1993;8:761–770 [CrossRef][PubMed]
    [Google Scholar]
  41. Nicolas P, Mäder U, Dervyn E, Rochat T, Leduc A et al. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science 2012;335:1103–1106 [CrossRef][PubMed]
    [Google Scholar]
  42. Bennett BD, Kimball EH, Gao M, Osterhout R, van Dien SJ et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 2009;5:593–599 [CrossRef][PubMed]
    [Google Scholar]
  43. Castro-Roa D, Garcia-Pino A, de Gieter S, van Nuland NAJ, Loris R et al. The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Nat Chem Biol 2013;9:811–817 [CrossRef][PubMed]
    [Google Scholar]
  44. Defeu Soufo HJ, Reimold C, Linne U, Knust T, Gescher J et al. Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein. Proc Natl Acad Sci USA 2010;107:3163–3168 [CrossRef][PubMed]
    [Google Scholar]
  45. Polekhina G, Thirup S, Kjeldgaard M, Nissen P, Lippmann C et al. Helix unwinding in the effector region of elongation factor EF-Tu-GDP. Structure 1996;4:1141–1151 [CrossRef][PubMed]
    [Google Scholar]
  46. Voorhees RM, Ramakrishnan V. Structural basis of the translational elongation cycle. Annu Rev Biochem 2013;82:203–236 [CrossRef][PubMed]
    [Google Scholar]
  47. van Noort JM, Kraal B, Sinjorgo KM, Persoon NL, Johanns ES et al. Methylation in vivo of elongation factor EF-Tu at lysine-56 decreases the rate of tRNA-dependent GTP hydrolysis. Eur J Biochem 1986;160:557–561 [CrossRef][PubMed]
    [Google Scholar]
  48. Pereira SF, Gonzalez RL, Dworkin J. Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor. Proc Natl Acad Sci USA 2015;112:E3274E3281 [CrossRef][PubMed]
    [Google Scholar]
  49. Nissen P, Thirup S, Kjeldgaard M, Nyborg J. The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA. Structure 1999;7:143–156 [CrossRef][PubMed]
    [Google Scholar]
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