Akasaka Kazuyuki, Maeno Akihiro, Yamazaki Akira
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan; Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan.
Laboratory of Chemistry, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan.
Biophys Chem. 2017 Dec;231:10-14. doi: 10.1016/j.bpc.2017.04.008. Epub 2017 Apr 19.
A bacterial spore protects itself with an unusually high concentration (~10% in dry weight of spore) of dipicolinic acid (DPA), the release of which is considered the crucial step for inactivating it under mild pressure and temperature conditions. However, the process of how the spore releases DPA in response to pressure remains obscure. Here we apply H high-resolution high-pressure NMR spectroscopy, for the first time, to the spore suspension of Bacillus subtilis natto and monitor directly and in real-time the leaking process of DPA in response to pressure of 200MPa at 20°C. We find that about one third of the total DPA leaks immediately upon applying pressure, but that the rest leaks slowly in hrs upon decreasing the pressure. Once DPA is fully released from the spore, the proteins of the spore become easily denatured at a mild temperature, e.g., 80°C, much below the temperature commonly used to inactivate spores (121°C). The success of the present experiment opens a new avenue for studying bacterial spores and cells at the molecular level in response to pressure, temperature and other perturbations.
细菌芽孢通过异常高浓度（约占芽孢干重的10%）的吡啶二羧酸（DPA）来保护自身，DPA的释放被认为是在温和的压力和温度条件下使其失活的关键步骤。然而，芽孢如何响应压力释放DPA的过程仍不清楚。在此，我们首次将高分辨率高压核磁共振光谱应用于纳豆芽孢杆菌的芽孢悬液，并在20℃下直接实时监测DPA在200MPa压力下的泄漏过程。我们发现，施加压力后，约三分之一的总DPA会立即泄漏，但其余的在减压后数小时内缓慢泄漏。一旦DPA从芽孢中完全释放，芽孢的蛋白质在温和温度（如80℃）下就很容易变性，该温度远低于通常用于使芽孢失活的温度（121℃）。本实验的成功为在分子水平上研究细菌芽孢和细胞对压力、温度及其他扰动的响应开辟了一条新途径。
