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Updated: Mar 19, 2026
Enriching Subcellular Proteins in Leptospira Using a Triton X-114-Based Fractionation Approach
Published on: August 8, 2025
Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans
Szilvia Barkó1, Dávid Szatmári1, Emőke Bódis1
1Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary.
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.
Insights
Researchers identified and characterized Li-MreB, a protein essential for the corkscrew shape of Leptospira interrogans. This discovery aids in understanding bacterial morphology and developing new methods for prokaryotic cation measurement.
Area of Science:
- Microbiology
- Bacterial Morphology
- Protein Biochemistry
Background:
- Weil's syndrome is caused by Leptospira interrogans, a Gram-negative bacterium with a unique corkscrew shape.
- The molecular basis for L. interrogans' distinctive morphology remains largely unknown.
- Bacterial cell wall synthesis is often regulated by MreB, an actin homolog.
Purpose of the Study:
- To identify and characterize the MreB protein from L. interrogans (Li-MreB).
- To investigate the polymerization properties of Li-MreB.
- To develop a method for measuring cation concentrations in prokaryotes.
Main Methods:
- Expressed and purified His-tagged Li-MreB in E. coli, requiring ATP for solubility.
- Used Alexa488 for fluorescent labeling of Li-MreB.
- Monitored Li-MreB assembly/disassembly using light scattering and sedimentation studies.
- Developed a method to measure monovalent cation concentrations in E. coli.
Main Results:
- Li-MreB polymerization is dependent on MgCl2 and KCl.
- Fluorescence spectroscopy revealed cation-induced activation and polymerization of Li-MreB.
- Purified Li-MreB formed filament bundles and sheets.
- A novel method for measuring prokaryotic cation concentrations was established.
Conclusions:
- Successfully identified and characterized the cation-dependent polymerization of a novel MreB from a non-rod shaped bacterium.
- Established a method for quantifying cation concentrations within prokaryotes.
- This work provides a foundation for future structural studies of Li-MreB.
