1887

Abstract

Era GTPase is universally present in microbes including (Mtb) complex bacteria. While Era is known to regulate ribosomal assembly in and predicted to be essential for growth, its function in mycobacteria remains obscured. Herein, we show that Era ortholog in the attenuated Mtb H37Ra strain, MRA_2388 (annotated as Era) is a cell envelope localized protein harbouring critical GTP-binding domains, which interacts with several envelope proteins of Mtb. The purified Era from (annotated as Era) exhibiting ~90 % sequence similarity with Era, exists in monomeric conformation. While it is co-purified with RNA upon overexpression in , the presence of RNA does not modulate the GTPase activity of the Era as against its counterpart from other organisms. CRISPRi silencing of does not show any substantial effect on the growth of Mtb H37Ra, which suggests a redundant function of Era in mycobacteria. Notably, no effect on ribosome assembly, protein synthesis or bacterial susceptibility to protein synthesis inhibitors was observed upon depletion of Era in Mtb H37Ra, further indicating a divergent role of Era GTPase in mycobacteria.

Funding
This study was supported by the:
  • Department of Biotechnology, Ministry of Science and Technology, India (Award BT/PR25690/GET/119/142/2017)
    • Principle Award Recipient: NisheethAgarwal
  • Council of Scientific & Industrial Research, India (Award 19/06/2016 (i) EU-V)
    • Principle Award Recipient: Pramilapal
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001200
2022-08-02
2024-12-02
Loading full text...

Full text loading...

/deliver/fulltext/micro/168/8/mic001200.html?itemId=/content/journal/micro/10.1099/mic.0.001200&mimeType=html&fmt=ahah

References

  1. Caldon CE, Yoong P, March PE. Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function. Mol Microbiol 2001; 41:289–297 [View Article] [PubMed]
    [Google Scholar]
  2. Agarwal N, Pareek M, Thakur P, Pathak V. Functional characterization of EngA(MS), a P-loop GTPase of Mycobacterium smegmatis. PLoS One 2012; 7:e34571 [View Article] [PubMed]
    [Google Scholar]
  3. March PE, Lerner CG, Ahnn J, Cui X, Inouye M. The Escherichia coli Ras-like protein (Era) has GTPase activity and is essential for cell growth. Oncogene 1988; 2:539–544 [PubMed]
    [Google Scholar]
  4. Ahnn J, March PE, Takiff HE, Inouye M. A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins. Proc Natl Acad Sci U S A 1986; 83:8849–8853 [View Article] [PubMed]
    [Google Scholar]
  5. Takiff HE, Chen SM, Court DL. Genetic analysis of the rnc operon of Escherichia coli. J Bacteriol 1989; 171:2581–2590 [View Article] [PubMed]
    [Google Scholar]
  6. Gollop N, March PE. A GTP-binding protein (Era) has an essential role in growth rate and cell cycle control in Escherichia coli. J Bacteriol 1991; 173:2265–2270 [View Article] [PubMed]
    [Google Scholar]
  7. Gollop N, March PE. Localization of the membrane binding sites of Era in Escherichia coli. Res Microbiol 1991; 142:301–307 [View Article] [PubMed]
    [Google Scholar]
  8. Lerner CG, Inouye M. Pleiotropic changes resulting from depletion of Era, an essential GTP-binding protein in Escherichia coli. Mol Microbiol 1991; 5:951–957 [View Article] [PubMed]
    [Google Scholar]
  9. Sayed A, Matsuyama S i, Inouye M. Era, an essential Escherichia coli small G-protein, binds to the 30S ribosomal subunit. Biochem Biophys Res Commun 1999; 264:51–54 [View Article] [PubMed]
    [Google Scholar]
  10. Hang JQ, Zhao G. Characterization of the 16S rRNA- and membrane-binding domains of Streptococcus pneumoniae Era GTPase: structural and functional implications. Eur J Biochem 2003; 270:4164–4172 [View Article] [PubMed]
    [Google Scholar]
  11. Tu C, Zhou X, Tropea JE, Austin BP, Waugh DS et al. Structure of ERA in complex with the 3’ end of 16S rRNA: implications for ribosome biogenesis. Proc Natl Acad Sci U S A 2009; 106:14843–14848 [View Article] [PubMed]
    [Google Scholar]
  12. Choudhary E, Thakur P, Pareek M, Agarwal N. Gene silencing by CRISPR interference in mycobacteria. Nat Commun 2015; 6:6267 [View Article] [PubMed]
    [Google Scholar]
  13. Thakur P, Gantasala NP, Choudhary E, Singh N, Abdin MZ et al. The preprotein translocase YidC controls respiratory metabolism in Mycobacterium tuberculosis. Sci Rep 2016; 6:24998 [View Article] [PubMed]
    [Google Scholar]
  14. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4:406–425 [View Article] [PubMed]
    [Google Scholar]
  15. Van Veldhoven PP, Mannaerts GP. Inorganic and organic phosphate measurements in the nanomolar range. Anal Biochem 1987; 161:45–48 [View Article] [PubMed]
    [Google Scholar]
  16. Schmidt EK, Clavarino G, Ceppi M, Pierre P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods 2009; 6:275–277 [View Article] [PubMed]
    [Google Scholar]
  17. Verstraeten N, Fauvart M, Versées W, Michiels J. The universally conserved prokaryotic GTPases. Microbiol Mol Biol Rev 2011; 75:507–542 [View Article] [PubMed]
    [Google Scholar]
  18. Britton RA, Powell BS, Dasgupta S, Sun Q, Margolin W et al. Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli. Mol Microbiol 1998; 27:739–750 [View Article] [PubMed]
    [Google Scholar]
  19. Minkovsky N, Zarimani A, Chary VK, Johnstone BH, Powell BS et al. Bex, the Bacillus subtilis homolog of the essential Escherichia coli GTPase Era, is required for normal cell division and spore formation. J Bacteriol 2002; 184:6389–6394 [View Article] [PubMed]
    [Google Scholar]
  20. Morimoto T, Loh PC, Hirai T, Asai K, Kobayashi K et al. Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis. Microbiology 2002; 148:3539–3552 [View Article] [PubMed]
    [Google Scholar]
  21. Matsuo Y, Morimoto T, Kuwano M, Loh PC, Oshima T et al. The GTP-binding protein YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. J Biol Chem 2006; 281:8110–8117 [View Article] [PubMed]
    [Google Scholar]
  22. Wood A, Irving SE, Bennison DJ, Corrigan RM. The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus. PLoS Genet 2019; 15:e1008346 [View Article] [PubMed]
    [Google Scholar]
  23. Hartzell PL. Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase. Proc Natl Acad Sci U S A 1997; 94:9881–9886 [View Article] [PubMed]
    [Google Scholar]
  24. Chen X, Court DL, Ji X. Crystal structure of ERA: a GTPase-dependent cell cycle regulator containing an RNA binding motif. Proc Natl Acad Sci U S A 1999; 96:8396–8401 [View Article] [PubMed]
    [Google Scholar]
  25. Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature 1991; 349:117–127 [View Article] [PubMed]
    [Google Scholar]
  26. Hwang J, Inouye M. An essential GTPase, der, containing double GTP-binding domains from Escherichia coli and Thermotoga maritima. J Biol Chem 2001; 276:31415–31421 [View Article] [PubMed]
    [Google Scholar]
  27. Karbstein K. Role of GTPases in ribosome assembly. Biopolymers 2007; 87:1–11 [View Article] [PubMed]
    [Google Scholar]
  28. Mishra R, Gara SK, Mishra S, Prakash B. Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: implications for GTP hydrolysis. Proteins 2005; 59:332–338 [View Article] [PubMed]
    [Google Scholar]
  29. Ruzheinikov SN, Das SK, Sedelnikova SE, Baker PJ, Artymiuk PJ et al. Analysis of the open and closed conformations of the GTP-binding protein YsxC from Bacillus subtilis. J Mol Biol 2004; 339:265–278 [View Article] [PubMed]
    [Google Scholar]
  30. Ojha A, Anand M, Bhatt A, Kremer L, Jacobs WR Jr et al. GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell 2005; 123:861–873 [View Article] [PubMed]
    [Google Scholar]
  31. DeJesus MA, Gerrick ER, Xu W, Park SW, Long JE et al. Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis. mBio 2017; 8:e02133-16 [View Article] [PubMed]
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.001200
Loading
/content/journal/micro/10.1099/mic.0.001200
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error