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首页> 外文期刊>Journal of bacteriology >Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth
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Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

机译：枯草芽孢杆菌孢子萌发蛋白复合物在孢子萌发和生长过程中的动力学分析

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Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ～50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ～2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.
机译：枯草芽孢杆菌孢子的萌发通常是当环境中的营养物质与孢子内膜（IM）中的发芽受体（GRs）相互作用时启动的，其中大多数脂质是不可移动的。 GRs和另一种萌发蛋白GerD在休眠孢子的IM中共定位在一个小的焦点上，称为“ germinosome”，这种共定位或焦点形成取决于GerD，这对于GR依赖的孢子快速萌发也是必不可少的。为了确定孢子萌发和生长过程中生殖小体和发芽蛋白的命运，我们采用了差分干涉显微镜和落射荧光显微镜，通过与发芽蛋白的荧光融合来追踪发芽孢子，并使用蛋白质印迹分析来测量发芽蛋白的水平。我们发现，在芽孢萌发后，发芽体病灶最终变为更大的分散模式，≥75％的孢子种群在发芽1 h的孢子中表现出这种模式，尽管> 80％萌发30分钟的孢子保留了发芽体病灶。蛋白质印迹分析表明，尽管在萌发的孢子中，GerD水平在15分钟内降低了约50％，但在这段时间内二吡啶甲酸释放所必需的GR蛋白和SpoVA蛋白的水平变化很小。由于发芽过程中发芽体的分散比GerD水平的降低要慢，因此发芽体的稳定性不会因GerD水平降低约2倍而受到损害，或者其他因素（例如快速IM脂质迁移的恢复）在发芽体中也很重要随着孢子萌发的进行而分散。

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