Pogliano K, Harry E, Losick R
Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138, USA.
Mol Microbiol. 1995 Nov;18(3):459-70. doi: 10.1111/j.1365-2958.1995.mmi_18030459.x.
We describe the application of immunofluorescence microscopy to visualization of the subcellular localization of proteins involved in coat morphogenesis and chromosome packaging during the process of sporulation in Bacillus subtilis. In confirmation and extension of previous findings, we show that SpolVA, which is responsible for guiding coat formation to the surface of the outer membrane that surrounds the developing spore, assembles into a shell that is located close to or on the surface of this enveloping membrane. CotE, which is responsible for the formation of the outer layer of the coat, assembles into a second shell of apparently larger diameter. Assembly of SpolVA could be detected as early as the morphological stage of polar septation and closely followed the enveloping membrane of the mother cell during the stage of engulfment, thereby providing a sensitive and diagnostic marker for this phagocytic-like process. Surprisingly, the chromosome of the developing spore and the small, acid-soluble proteins, known as alpha/beta-type SASPs, that are known to coat the spore chromosome, were found to co-localize to a doughnut-like ring of approximately 1 micrometer in diameter. The use of a double mutant lacking the alpha/beta-type SASP demonstrated that these high abundance, DNA-binding proteins are responsible for packaging the chromosome of the developing spore into this unusual structure. We conclude that sporulation in B. subtilis is a fertile system for addressing cell biological problems in a bacterium and that immunofluorescence microscopy provides a sensitive method for visualizing protein subcellular localization at high resolution.
我们描述了免疫荧光显微镜在可视化枯草芽孢杆菌孢子形成过程中参与被膜形态发生和染色体包装的蛋白质亚细胞定位方面的应用。在证实和扩展先前研究结果的过程中，我们发现，负责将被膜形成引导至围绕发育中孢子的外膜表面的SpolVA组装成一个位于该包被膜表面附近或之上的壳层。负责被膜外层形成的CotE组装成一个直径明显更大的第二壳层。早在极性隔膜的形态学阶段就能检测到SpolVA的组装，并且在吞噬阶段紧密跟随母细胞的包被膜，从而为这种类似吞噬作用的过程提供了一个灵敏的诊断标记。令人惊讶的是，发现发育中孢子的染色体以及已知包裹孢子染色体的小的酸溶性蛋白质（称为α/β型小酸溶性芽孢蛋白，SASPs）共定位于一个直径约1微米的甜甜圈状环中。使用缺乏α/β型SASP的双突变体表明，这些高丰度的DNA结合蛋白负责将发育中孢子的染色体包装成这种不寻常的结构。我们得出结论，枯草芽孢杆菌的孢子形成是解决细菌细胞生物学问题的一个丰富系统，并且免疫荧光显微镜为高分辨率可视化蛋白质亚细胞定位提供了一种灵敏的方法。
