The Library

The pH dependence of polymerization and bundling by the essential bacterial cytoskeltal protein FtsZ

Tools

Pacheco-Gómez, Raúl, Roper, David I., Dafforn, Tim and Rodger, Alison. (2011) The pH dependence of polymerization and bundling by the essential bacterial cytoskeltal protein FtsZ. PLoS One, Vol.6 (No.6). e19369. ISSN 1932-6203

Preview

PDF
WRAP_Pacheco_Gomez_pH_dependence.pdf - Published Version - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
Available under License Creative Commons Attribution.
Download (3276Kb)

Official URL: http://dx.doi.org/10.1371/journal.pone.0019369

Abstract

There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution-it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)-it stablizes associations of protofilaments.

Item Type:	Journal Article
Subjects:	Q Science > QR Microbiology
Divisions:	Faculty of Science > Chemistry Faculty of Science > Life Sciences (2010- ) Faculty of Science > Molecular Organisation and Assembly in Cells (MOAC)
Library of Congress Subject Headings (LCSH):	Polymerization, Escherichia coli -- Physiology, Cell division
Journal or Publication Title:	PLoS One
Publisher:	Public Library of Science
ISSN:	1932-6203
Official Date:	28 June 2011
Volume:	Vol.6
Number:	No.6
Page Range:	e19369
Identification Number:	10.1371/journal.pone.0019369
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
Funder:	Engineering and Physical Sciences Research Council (EPSRC)
Grant number:	GR/S47113/01 (EPSRC), GR/T09224/01 (EPSRC)
References:	1. Adams DW, Errington J (2009) Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nat Rev Microbiol 7: 642–653. 2. Errington J, Daniel RA, Scheffers DJ (2003) Cytokinesis in bacteria. Microbiol Mol Biol Rev 67: 52–65, table of contents. 3. Bramhill D, Thompson CM (1994) GTP-dependent polymerisation of Escherichia coli FTsZ protein to form tubules. Proc Natl Acad Sci 91: 5813–5817. 4. Mukherjee A, Lutkenhaus J (1994) Guanine nucleotide-dependent assembly of FtsZ into filaments. Journal of Bacteriology 176: 2754–2758. 5. Stricker J, Maddox P, Salmon ED, Erickson HP (2002) Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching. Proc Natl Acad Sci 99: 3171–3175. 6. Haydon DJ, Stokes NR, Ure R, Galbraith G, Bennett JM, et al. (2008) An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science 321: 1673–1675. 7. Chen Y, Erickson HP (2009) FtsZ Filament Dynamics at Steady State: Subunit Exchange with and without Nucleotide Hydrolysis. Biochemistry 48: 6664–6673. 8. Marrington R, Small E, Rodger A, Dafforn TR, Addinall S (2004) FtsZ fibre bundling is triggered by a calcium-induced conformational change in bound GTP. J Biol Chem 279: 48821–48829. 9. Small E, Marrington R, Rodger A, Scott DJ, K. S, et al. (2007) FtsZ polymerbundling by the Escherichia coli ZapA orthologue, YgfE involves a conformational change in bound GTP. J Mol Biol 369: 211–221. 10. Wilks JC, Slonczewski JL (2007) pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry. J Bacteriol 189: 5601–5607. 11. Mukherjee A, Lutkenhaus J (1998) Dynamic assembly of FtsZ regulated by GTP hydrolysis. EMBO Journal 17: 462–469. 12. Scheffers D-J (2008) The effect of MinC on FtsZ polymerization is pH dependent and can be counteracted by ZapA. FEBS Lett 582: 2601–2608. 13. Mendieta J, Rico AI, Lo´pez-Vin˜as E, Vicente M, Mingorance J, et al. (2009) Structural and Functional Model for Ionic (K+/Na+) and pH Dependence of GTPase Activity and Polymerization of FtsZ, the Prokaryotic Ortholog of Tubulin. J Mol Biol 390: 17–25. 14. Bradford M (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem 72: 248–254. 15. Mukherjee A, Lutkenhaus J (1998) Purification, Assembly and Localization of FtsZ. Methods on Enzymology 298: 296–305. 16. Small E, Addinall SG (2003) Dynamic FtsZ polymerization is sensitive to the GTP to GDP ratio and can be maintained at steady state using a GTPregeneration system. Microbiology 149. 17. Mukherjee AaL, J. (1999) Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations. Journal of Bacteriology 181: 823–832. 18. Marrington R, Dafforn TR, Halsall DJ, Macdonald JI, Hicks M, et al. (2005) Validation of new microvolume Couette linear dichroism cells. Analyst. pp 1608–1616. 19. Marrington R (2004) Polarised spectroscopy of biomacromolecules. Coventry: Warwick University. 20. Nordh J, Deinum J, Norden B (1986) Flow orientation of brain microtubules studied by linear dichroism. European Biophysical Journal 14: 113–122. 21. Marrington R, Seymour M, Rodger A (2006) A new method for fibrous protein analysis illustrated by application to tubulin microtubule polymerisation and depolymerisation. Chirality 18: 680–690. 22. Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. In: Biochemistry, ed. Proc Natl Acad Sci, USA. pp 4884–4887. 23. Johnson WC (1999) Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins Struct Funct Genet 35: 307–312. 24. Johnson WC, (Jr) (1988) Secondary structure of proteins through circular dichroism spectroscopy. Ann Rev Biophys Biophys Chem 17: 145–166. 25. Rodger A, Rajendra J, Marrington R, Ardhamar M, Norden B, et al. (2002) Membranes and proteins. Phys Chem Chem Phys 4: 4051–4057. 26. Norde´n B, Rodger A, Dafforn TR (2010) Linear dichroism and circular dichroism: a textbook on polarized spectroscopy. Cambridge: Royal Society of Chemistry. 304 p. 27. Marrington R, Addinall S, Dafforn TR, Small E, Rodger A (2004) Calcium induced conformational change in GTP bound to FtsZ: A trigger for fibre bundling. In Progress. 28. Erickson HP (2009) Modeling the physics of FtsZ assembly and force generation. Proc Nat Acad Sci. pp 9238–9243. 29. Mingorance J, Rueda S, Gomez-Puertas P, Valencia A, Vicente M (2001) Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover. Molecular Microbiology 41: 83–91. 30. Lu C, Reedy M, Erickson HP (2000) Straight and curved conformations of FtsZ are regulated by GTP hydrolysis. J Bacteriol 182: 164–170.
URI:	http://wrap.warwick.ac.uk/id/eprint/38804