Wahome Paul G, Cowan Ann E, Setlow Barbara, Setlow Peter
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3305, USA.
Arch Microbiol. 2009 May;191(5):403-14. doi: 10.1007/s00203-009-0465-z. Epub 2009 Feb 28.
Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock > or =0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is partially dependent on three likely MS channel proteins of small conductance (MscS), YfkC, YhdY, and YkuT. Bacillus subtilis MS proteins were fused with green fluorescent protein (GFP) at their C termini; at least the MscL-, YfkC-, and YkuT-GFP fusions were functional and overexpression of YkuT-GFP, or YkuT alone abolished log phase mscL cells' osmotic downshock sensitivity. Western blot analysis found high levels of MscL-GFP in early exponential phase cells with levels subsequently decreasing greatly. MscS-GFP proteins were present in exponential phase cells, but again disappeared almost completely in stationary phase cells and these proteins were not detected in spores. Western blot analyses further showed that MS-GFP proteins were associated with the plasma membrane, as expected. Fluorescence microscopy confirmed the localization of MscL-GFP and YhdY-GFP to the plasma membrane, with non-uniform distribution of these proteins along this membrane consistent with but by no means proving that these proteins are present in a helical array.
缺乏编码大电导机械敏感（MS）通道的mscL基因的对数期枯草芽孢杆菌细胞对≥0.5M的渗透压下降敏感。然而，枯草芽孢杆菌mscL细胞在对数后期和稳定期早期生长过程中产生了渗透压下降抗性，这部分依赖于三种可能的小电导MS通道蛋白（MscS）、YfkC、YhdY和YkuT。枯草芽孢杆菌的MS蛋白在其C末端与绿色荧光蛋白（GFP）融合；至少MscL-、YfkC-和YkuT-GFP融合蛋白是有功能的，YkuT-GFP或单独的YkuT过表达消除了对数期mscL细胞的渗透压下降敏感性。蛋白质免疫印迹分析发现，在指数生长期早期细胞中MscL-GFP水平很高，随后水平大幅下降。MscS-GFP蛋白存在于指数生长期细胞中，但在稳定期细胞中几乎完全消失，并且在孢子中未检测到这些蛋白。蛋白质免疫印迹分析进一步表明，正如预期的那样，MS-GFP蛋白与质膜相关。荧光显微镜证实了MscL-GFP和YhdY-GFP定位于质膜，这些蛋白沿该膜的分布不均匀，这与但绝不能证明这些蛋白以螺旋阵列形式存在一致。
