Centre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität Marburg, Marburg, 35032, Germany.
BRAIN Biotech AG, Darmstädter Str. 34-36, Zwingenberg, 64673, Germany.
BMC Biol. 2023 Oct 4;21(1):206. doi: 10.1186/s12915-023-01684-1.
Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis.
We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system.
Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.
尽管跨细胞膜的蛋白质转运已经得到了深入研究，但在革兰氏阳性菌中，蛋白质如何穿过细胞壁仍然不清楚，革兰氏阳性菌被广泛应用于工业生产。我们研究了两种不同的芽孢杆菌（枯草芽孢杆菌和地衣芽孢杆菌）中α-淀粉酶 AmyE 的分泌。
我们发现，AmyE 的 C 端融合荧光报告蛋白 mCherry 后通过离散的斑点进行分泌，这些斑点显示出非常低的动态性。这些斑点在细胞壁的许多位置可见，持续数分钟。只有在高拷贝数质粒表达时，才能观察到我们称之为“分泌区”的结构。这些区域与细胞表面可见的 AmyE 活性相对应，表明它们释放了活性酶。它们与 SecA 信号重叠，但并不经常与分泌 ATP 酶共定位。单颗粒跟踪显示，在细胞膜上，SecA 和参与 AmyE 分泌的 SecDF 的动态性高于 AmyE。这些实验表明，SecA 最初将 AmyE 分子穿过细胞膜转运，然后扩散到不同的转运体。SecA 的单分子跟踪表明，SecA 存在三种不同的扩散状态，在 AmyE 过表达时会发生变化，但增加 AmyE 分泌似乎不会使系统过载。
因为只有在进入和处于静止期的过渡阶段才能发现分泌区，所以扩散而不是基于从内到外的细胞壁生长的被动运输可能会释放出 AmyE，因此可能会释放出一般的分泌蛋白。我们的研究结果表明，在芽孢杆菌属的细菌中，至少对于过表达的蛋白质，存在主动跨细胞膜转运和缓慢的、被动的穿过细胞壁的过程。
