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

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.

FASCIN and alpha-actinin can regulate the conformation of actin filaments.

BACKGROUND: Actin filament bundling proteins mediate numerous processes in cells such as the formation of cell membrane protrusions or cell adhesions and stress fiber based locomotion. Among them alpha-actinin and fascin are the most abundant ones. This work characterizes differences in molecular motions in actin filaments due to the binding of these two actin bundling proteins. METHODS: We investigated how alpha-actinin and fascin binding modify the conformation of actin filaments by using conventional and saturation transfer EPR methods. RESULTS: The result characteristic for motions on the microsecond time scale showed that both actin bundling proteins made the bending and torsional twisting of the actin filaments slower. When nanosecond time scale molecular motions were described the two proteins were found to induce opposite changes in the actin filaments. The binding of one molecule of alpha-actinin or fascin modified the conformation of numerous actin protomers. CONCLUSION: As fascin and alpha-actinin participates in different cellular processes their binding can serve the proper tuning of the structure of actin by establishing the right conformation for the interactions with other actin binding proteins. Our observations are in correlation with the model where actin filaments fulfill their biological functions under the regulation by actin-binding proteins. GENERAL SIGNIFICANCE: Supporting the general model for the cellular regulation of the actin cytoskeleton we showed that two abundant actin bundling proteins, fascin and alpha-actinin, alter the conformation of actin filaments through long range allosteric interactions in two different ways providing the structural framework for the adaptation to specific biological functions.

tert-Butyl hydroperoxide-induced differing plasma membrane and oxidative stress processes in yeast strains BY4741 and erg5Δ.

The molecular mechanism of tert-butyl hydroperoxide (t-BuOOH) elicited cytotoxicity and the background of t-BuOOH sensitivity were studied in the Saccharomyces cerevisiae ergosterol-less gene deletion mutant erg5Δ and its parental strain BY4741. In comparison to BY4741, untreated erg5Δ cells exhibited alterations in sterol and fatty acid compositions of the plasma membrane, as reflected by the inherent amphotericin B resistance, an elevated level (31%) of plasma membrane rigidity and a decreased uptake of glycerol. Surprisingly, the untreated erg5Δ cells exhibited an unbalanced intracellular redox state, accompanied by the continuous upregulation of the antioxidant enzymes Mn superoxide dismutase, catalase, and glutathione S-transferase, which resulted in decreased specific concentrations of superoxide and peroxides and elevated levels of the hydroxyl radical and thiols. The 2.5-fold sensitivity of erg5Δ to t-BuOOH suggested that the oxidative stress adaptation processes of the mutant could not restore the redox homeostasis of the cells and there is an overlap between sterol and redox homeostases. t-BuOOH treatment of both strains induced adaptive modification of the sterol and fatty acid compositions, increased the plasma membrane fluidity and elevated the specific activities of most antioxidant enzymes through specific regulation processes in a strain-dependent manner.