Department of Biophysics, Medical School, University of Pécs, Pécs, Hungary.
Department of Medical Microbiology and Immunology, Medical School, University of Pécs, Pécs, Hungary.
Sci Rep. 2020 Jul 20;10(1):12002. doi: 10.1038/s41598-020-68960-w.
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.
在这里，我们测量了培养的革兰氏阴性和革兰氏阳性细菌中的几种离子浓度，并分析了它们对肌动蛋白同源物 MreB 形成聚合物的影响。我们测量了钩端螺旋体、枯草芽孢杆菌和大肠杆菌中的钾、钠、氯、钙和镁离子浓度。这些离子在细胞内产生的离子强度在 130-273 mM 范围内变化。细胞内钠离子浓度范围在 122 和 296 mM 之间，钾离子浓度范围在 5 和 38 mM 之间。然而，这些水平受到细胞外离子水平的显著影响。使用一种新的过滤方法，从大肠杆菌中异源表达和纯化了钩端螺旋体、立克次体和大肠杆菌 MreB，以制备 MreB 聚合物。通过共焦显微镜研究了在不同离子强度条件下，Alexa-488 标记的 MreB 聚合物的结构和稳定性，并通过测量光散射评估了 MreB 聚合速率。在 200-300 mM 的单离子浓度范围内，MreB 聚合最快。在该浓度范围内，MreB 纤维表现出高度的稳定性，并形成大的带状束组装体，在更高的离子强度下转化为广泛的薄片。将钙离子浓度从 0.2 变为 0 mM，然后变为 2 mM，会初始化 MreB 聚合物的快速重塑。
