Wang Shiwei, Brunt Jason, Peck Michael W, Setlow Peter, Li Yong-Qing
School of Chemical Engineering and Energy Technology, Dongguan University of Technology, Dongguan, China.
Gut Health and Food Safety, Quadram Institute Bioscience, Norwich, United Kingdom.
Front Microbiol. 2017 Oct 25;8:2047. doi: 10.3389/fmicb.2017.02047. eCollection 2017.
The Gram-positive spore-forming anaerobe is a significant cause of food spoilage, and it is also used as a surrogate for spores for testing the efficacy of commercial sterilization. spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual wild-type and germination mutant spores. Most individual spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at , all transitioned to rapid CaDPA release at , completed CaDPA release at , and finished peptidoglycan cortex hydrolysis at . , , , and times for individual spores were heterogeneous, but Δ ( - ) periods were relatively constant. However, variability in (or ) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After , some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25-30% at prior to . This has not been seen with spores of other species. Almost all spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a in L-alanine germination. Sublethal heat treatment potentiated spore germination with L-alanine, primarily by shortening T times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in spore germination.
革兰氏阳性产芽孢厌氧菌是食品变质的重要原因，它也被用作测试商业灭菌效果的孢子替代物。孢子还被提议作为将药物递送至肿瘤细胞用于癌症治疗的载体。孢子的这种应用需要它们萌发并恢复活性。在本研究中，使用拉曼光谱和微分干涉对比（DIC）显微镜分析多个单个野生型和萌发突变体孢子的萌发动力学。大多数用L-丙氨酸萌发的单个孢子在 时开始缓慢泄漏其约5%的大量钙吡啶二羧酸（CaDPA）储存库，在 时全部转变为快速CaDPA释放，在 时完成CaDPA释放，并在 时完成肽聚糖皮层水解。单个孢子的 、 、 和 时间是异质的，但Δ（ - ）时间段相对恒定。然而， （或 ）时间的变异性似乎是单个孢子萌发时间异质性的主要原因。在 之后，一些孢子在 之前第二次明显的DIC图像强度下降25 - 30%开始之前，DIC图像强度变化率也出现了另一个滞后。这在其他物种的孢子中未见。几乎所有缺乏皮层裂解酶（CLE）CwlJ的孢子在L-丙氨酸萌发中表现出 。亚致死热处理增强了孢子在L-丙氨酸中的萌发，主要是通过缩短 时间。缺乏CLEs SleB或CwlJ的孢子在L-丙氨酸中的萌发大大减慢，但缺乏所有萌发受体蛋白的孢子不能用L-丙氨酸萌发。这些各种萌发蛋白的缺失也降低了但并未消除与非GR依赖性萌发剂十二烷基胺和CaDPA的萌发，但没有CwlJ的孢子在CaDPA存在下不萌发。最后，孢子在萌发中表现出承诺，但在GR依赖性萌发中的记忆很小，且小于孢子萌发中的记忆。
