Solubility and Thermal Stability of Thermotoga maritima MreB.

The basis of MreB research is the study of the MreB protein from the Thermotoga maritima species, since it was the first one whose crystal structure was described. Since MreB proteins from different bacterial species show different polymerisation properties in terms of nucleotide and salt dependence, we conducted our research in this direction. For this, we performed measurements based on tryptophan emission, which were supplemented with temperature-dependent and chemical denaturation experiments. The role of nucleotide binding was studied through the fluorescent analogue TNP-ATP. These experiments show that Thermotoga maritima MreB is stabilised in the presence of low salt buffer and ATP. In the course of our work, we developed a new expression and purification procedure that allows us to obtain a large amount of pure, functional protein.

Author Correction: Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies.

Here, we measured the concentrations of several ions in cultivated Gram-negative and Gram-positive bacteria, and analyzed their effects on polymer formation by the actin homologue MreB. We measured potassium, sodium, chloride, calcium and magnesium ion concentrations in Leptospira interrogans, Bacillus subtilis and Escherichia coli. Intracellular ionic strength contributed from these ions varied within the 130-273 mM range. The intracellular sodium ion concentration range was between 122 and 296 mM and the potassium ion concentration range was 5 and 38 mM. However, the levels were significantly influenced by extracellular ion levels. L. interrogans, Rickettsia rickettsii and E. coli MreBs were heterologously expressed and purified from E. coli using a novel filtration method to prepare MreB polymers. The structures and stability of Alexa-488 labeled MreB polymers, under varying ionic strength conditions, were investigated by confocal microscopy and MreB polymerization rates were assessed by measuring light scattering. MreB polymerization was fastest in the presence of monovalent cations in the 200-300 mM range. MreB filaments showed high stability in this concentration range and formed large assemblies of tape-like bundles that transformed to extensive sheets at higher ionic strengths. Changing the calcium concentration from 0.2 to 0 mM and then to 2 mM initialized rapid remodelling of MreB polymers.