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



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.



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.



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.



This initial characterization of Li-MreB will enable future structural determination of the MreB filament from this corkscrew-shaped bacterium.
Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
Szilvia
S
Barkó
Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
Dávid
D
Szatmári
Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
Emőke
E
Bódis
Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
Katalin
K
Türmer
Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
Zoltán
Z
Ujfalusi
Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis 138673, Singapore.
David
D
Popp
Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis 138673, Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore.
Robert C
RC
Robinson
Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary; MTA-PTE Nuclear-Mitochondrial Interactions Research Group, Szigeti str. 12, Pécs H-7624, Hungary.
Miklós
M
Nyitrai
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D004926:Escherichia coli; D016680:Genome, Bacterial / Q000235:genetics; D007921:Leptospira interrogans / Q000235:genetics / Q000378:metabolism*; D007922:Leptospirosis / Q000382:microbiology; D008856:Microscopy, Fluorescence / Q000379:methods; D009711:Nucleotides / Q000378:metabolism; D058105:Polymerization
D000199:Actins; D001426:Bacterial Proteins; D002412:Cations; D009711:Nucleotides; D000255:Adenosine Triphosphate
Bacterial actin; Cytoskeleton; Fluorescence microscopy; MreB; Polymerization