Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans

Szilvia Barkó, Dávid Szatmári, Emoke Bódis, Katalin Türmer, Zoltán Ujfalusi, David Popp, Robert C. Robinson, Miklós Nyitrai

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Background Weil's syndrome is caused by Leptospira interrogans infections, a Gram negative bacterium with a distinct thin corkscrew cell shape. The molecular basis for this unusual morphology is unknown. In many bacteria, cell wall synthesis is orchestrated by the actin homolog, MreB. Methods Here we have identified the MreB within the L. interrogans genome and expressed the His-tagged protein product of the synthesized gene (Li-MreB) in Escherichia coli. Li-MreB did not purify under standard nucleotide-free conditions used for MreBs from other species, requiring the continual presence of ATP to remain soluble. Covalent modification of Li-MreB free thiols with Alexa488 produced a fluorescent version of Li-MreB. Results We developed native and denaturing/refolding purification schemes for Li-MreB. The purified product was shown to assemble and disassemble in MgCl2 and KCl dependent manners, as monitored by light scattering and sedimentation studies. The fluorescence spectrum of labeled Li-MreB-Alexa488 showed cation-induced changes in line with an activation process followed by a polymerization phase. The resulting filaments appeared as bundles and sheets under the fluorescence microscope. Finally, since the Li-MreB polymerization was cation dependent, we developed a simple method to measure monovalent cation concentrations within a test case prokaryote, E. coli. Conclusions We have identified and initially characterized the cation-dependent polymerization properties of a novel MreB from a non-rod shaped bacterium and developed a method to measure cation concentrations within prokaryotes. General significance. This initial characterization of Li-MreB will enable future structural determination of the MreB filament from this corkscrew-shaped bacterium.

Original languageEnglish
Pages (from-to)1942-1952
Number of pages11
JournalBiochimica et Biophysica Acta - General Subjects
Volume1860
Issue number9
DOIs
Publication statusPublished - Sep 1 2016
Externally publishedYes

Keywords

  • Bacterial actin
  • Cytoskeleton
  • Fluorescence microscopy
  • MreB
  • Polymerization

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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