Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae

Abstract

Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: The PASTA4 repeat is critical for pneumococcal cell division.
Fig. 2: Deletion of PASTA1, 2 and 3 repeats inactivates StkP and renders pneumococcal cell division independent of StkP kinase activity.
Fig. 3: PASTA1, 2 and 3 are equivalent and required for StkP activation independently from their cell-wall binding specificity.
Fig. 4: PASTA4 localizes StkP at the division septum.
Fig. 5: Taxonomic distribution and organization of PASTA repeats in Streptococcaceae and structural organization of PASTA4 and its properties for cell division.
Fig. 6: PASTA4 positions LytB to the division septum while PASTA123 repeats rule the thickness of the septal cell wall.

References

  1. Dworkin, J. Ser/Thr phosphorylation as a regulatory mechanism in bacteria. Curr. Opin. Microbiol. 24, 47–52 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mijakovic, I., Grangeasse, C. & Turgay, K. Exploring the diversity of protein modifications: special bacterial phosphorylation systems. FEMS Microbiol. Rev. 40, 398–417 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Manuse, S., Fleurie, A., Zucchini, L., Lesterlin, C. & Grangeasse, C. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis. FEMS Microbiol. Rev. 40, 41–56 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Pereira, S. F., Goss, L. & Dworkin, J. Eukaryote-like serine/threonine kinases and phosphatases in bacteria. Microbiol. Mol. Biol. Rev. 75, 192–212 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Yeats, C., Finn, R. D. & Bateman, A. The PASTA domain: a beta-lactam-binding domain. Trends Biochem. Sci. 27, 438 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Calvanese, L., Falcigno, L., Squeglia, F., D Auria G. & Berisio, R. PASTA in penicillin binding proteins and serine/threonine kinases: a recipe of structural, dynamic and binding properties. Curr. Med. Chem. https://doi.org/10.2174/0929867324666170216112746 (2017).

  7. Barthe, P., Mukamolova, G. V., Roumestand, C. & Cohen-Gonsaud, M. The structure of PknB extracellular PASTA domain from Mycobacterium tuberculosis suggests a ligand-dependent kinase activation. Structure 18, 606–615 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Maestro, B. et al. Recognition of peptidoglycan and beta-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae. FEBS Lett. 585, 357–363 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Squeglia, F. et al. Chemical basis of peptidoglycan discrimination by PrkC, a key kinase involved in bacterial resuscitation from dormancy. J. Am. Chem. Soc. 133, 20676–20679 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Mir, M. et al. The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization. PLoS Pathog. 7, e1002182 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Shah, I. M., Laaberki, M. H., Popham, D. L. & Dworkin, J. A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments. Cell 135, 486–496 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Calvanese, L. et al. Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis. Biopolymers 101, 712–719 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Morlot, C. et al. Interaction of penicillin-binding protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae R6 morphogenesis. Mol. Microbiol. 90, 88–102 (2013).

    CAS  PubMed  Google Scholar 

  14. Prigozhin, D. M. et al. Structural and genetic analyses of the Mycobacterium tuberculosis protein kinase B sensor domain identify a potential ligand-binding site. J. Biol. Chem. 291, 22961–22969 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hardt, P. et al. The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus. Int. J. Med. Microbiol. 307, 1–10 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Fleurie, A. et al. Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae. Mol. Microbiol. 83, 746–758 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Beilharz, K. et al. Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP. Proc. Natl Acad. Sci. USA 109, E905–E913 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Fleurie, A. et al. Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division. PLoS Genet. 10, e1004275 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Fleurie, A. et al. MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae. Nature 516, 259–262 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ulrych, A. et al. Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag. BMC Microbiol. 16, 247 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Stamsas, G. A. et al. Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae. Mol. Microbiol. 105, 954–967 (2017).

    Article  CAS  PubMed  Google Scholar 

  22. Zheng, J. J., Perez, A. J., Tsui, H. T., Massidda, O. & Winkler, M. E. Absence of the KhpA and KhpB (JAG/EloR) RNA-binding proteins suppresses the requirement for PBP2b by overproduction of FtsA in Streptococcus pneumoniae D39. Mol. Microbiol. https://doi.org/10.1111/mmi.13847 (2017).

    Google Scholar 

  23. Shaik, M. M., Maccagni, A., Tourcier, G., Di Guilmi, A. M. & Dessen, A. Structural basis of pilus anchoring by the ancillary pilin RrgC of Streptococcus pneumoniae. J. Biol. Chem. 289, 16988–16997 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Paracuellos, P. et al. The extended conformation of the 2.9-A crystal structure of the three-PASTA domain of a Ser/Thr kinase from the human pathogen Staphylococcus aureus. J. Mol. Biol. 404, 847–858 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Garcia, P., Gonzalez, M. P., Garcia, E., Lopez, R. & Garcia, J. L. LytB, a novel pneumococcal murein hydrolase essential for cell separation. Mol. Microbiol. 31, 1275–1281 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Rico-Lastres, P. et al. Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence. Sci. Rep. 5, 16198 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Berg, K. H., Biornstad, T. J., Straume, D. & Havarstein, L. S. Peptide-regulated gene depletion system developed for use in Streptococcus pneumoniae. J. Bacteriol. 193, 5207–5215 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chawla, Y. et al. Protein kinase B (PknB) of Mycobacterium tuberculosis is essential for growth of the pathogen in vitro as well as for survival within the host. J. Biol. Chem. 289, 13858–13875 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Turapov, O. et al. The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth. Open Biol. 5, 150025 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Ruggiero, A. et al. X-ray structural studies of the entire extracellular region of the serine/threonine kinase PrkC from Staphylococcus aureus. Biochem. J. 435, 33–41 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Gordon, E., Mouz, N., Duee, E. & Dideberg, O. The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance. J. Mol. Biol. 299, 477–485 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Canova, M. J., Kremer, L. & Molle, V. pETPhos: a customized expression vector designed for further characterization of Ser/Thr/Tyr protein kinases and their substrates. Plasmid 60, 149–153 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Cortay, J. C. et al. In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis. J. Biol. Chem. 269, 14885–14891 (1994).

    CAS  PubMed  Google Scholar 

  34. Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl. Environ. Microbiol. 67, 5190–5196 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ducret, A., Quardokus, E. M. & Brun, Y. V. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat. Microbiol. 1, 16077 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Adams, P. D. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Hayashi, K. A rapid determination of sodium dodecyl sulfate with methylene blue. Anal. Biochem. 67, 503–506 (1975).

    Article  CAS  PubMed  Google Scholar 

  42. Fenton, A. K., Mortaji, L. E., Lau, D. T., Rudner, D. Z. & Bernhardt, T. G. CozE is a member of the MreCD complex that directs cell elongation in Streptococcus pneumoniae. Nat. Microbiol. 2, 16237 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  43. De Las Rivas, B., Garcia, J. L., Lopez, R. & Garcia, P. Purification and polar localization of pneumococcal LytB, a putative endo-beta-N-acetylglucosaminidase: the chain-dispersing murein hydrolase. J. Bacteriol. 184, 4988–5000 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Eddy, S. R. A new generation of homology search tools based on probabilistic inference. Genome Inform. 23, 205–211 (2009).

    PubMed  Google Scholar 

  45. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Criscuolo, A. & Gribaldo, S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 10, 210 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Jauffrit, F. et al. RiboDB database: a comprehensive resource for prokaryotic systematics. Mol. Biol. Evol. 33, 2170–2172 (2016).

    Article  CAS  PubMed  Google Scholar 

  49. Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Centre National de la Recherche Scientifique, Université Claude Bernard Lyon 1, Agence Nationale de la Recherche (ANR-12-BSV3-0008-01 and ANR-15-CE32-0001-01), Region Auvergne-Rhône-Alpes (financial support for L.Z., C.M. and P.S.G.) and Bettencourt Schueller Foundation. We thank A. Ducret for implementing the heat maps and violin plots in MicrobeJ and carefully reading the paper. We thank P. Garcia at the Centro de Investigaciones Biológicas for providing the plasmid encoding GFP–LytB and the anti-LytB antibody, and L. S. Havarstein at the Norwegian University of Life Sciences for providing S. pneumoniae H130 genomic DNA. We acknowledge the contribution of the ‘Protein Science’ and ‘Plateau Technique Imagerie/Microscopie’ microscopy facilities of the Structure Fédérative de Recherche Biosciences Gerland-Lyon Sud (UMS344/US8), the ‘Centre Technologique des Microstructures’ of the Université Claude Bernard Lyon I, and Swiss Lightsource and European Synchrotron Radiation Facility synchrotron beamlines.

Author information

Authors and Affiliations

Authors

Contributions

L.Z., C.M. and C.F. conducted the cell biology and genetics experiments, purified proteins for the surface plasmon resonance experiments and performed the western blot analysis. F.G., P.G. and V.G.-C. conducted the crystallogenesis experiments and solved the structure of PASTA4. C.C. performed the transmission electron microscopy. P.S.G. and C.B-A. performed the phylogeny analyses. L.Z., C.M., P.S.G., C.C., V.G.-C., F.G., C.F. and S.G. designed and analysed the data with assistance from C.B.-A., P.G. and C.G. C.G. wrote the paper. All authors edited the paper.

Corresponding author

Correspondence to Christophe Grangeasse.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Correspondence and requests for materials should be addressed to C.G.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–11 and Supplementary Tables 1–7

Life Sciences Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zucchini, L., Mercy, C., Garcia, P.S. et al. PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae . Nat Microbiol 3, 197–209 (2018). https://doi.org/10.1038/s41564-017-0069-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-017-0069-3

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing