Biochimica et biophysica acta · 2016
Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans.
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Barkó Szilvia, Szatmári Dávid, Bódis Emőke, Türmer Katalin, Ujfalusi Zoltán, Popp David, Robinson Robert C, Nyitrai Miklós
Аннотация
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.
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.