Ducret Adrien, Théodoly Olivier, Mignot Tâm
Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Université Aix-Marseille, Marseille, France.
Methods Mol Biol. 2013;966:97-107. doi: 10.1007/978-1-62703-245-2_6.
A large number of bacterial species move smoothly on solid surfaces in the absence of extracellular -organelles. In the deltaproteobacterium Myxococcus xanthus, this surface motion, termed gliding motility, involves a novel macromolecular machinery Agl-Glt. During the motility process, the Agl-Glt system, an integral envelope protein complex, is assembled on the ventral side of the cell. Doing so, the complex couples surface adhesion to the activity of the Agl motility motor. On the cytosolic side, the Agl-Glt system is linked to the bacterial actin cytoskeleton MreB. It is proposed that motility is produced when surface immobilized Agl-Glt complexes produce traction on a rigid track, possibly the MreB cables. Testing this hypothesis directly requires both microfluidic techniques to perturb the motility process with inhibitors (i.e., A22, CCCP) and state-of-the-art microscopy techniques (i.e., TIRF and AFM). These approaches require a microscopy chamber where the cells glide in liquid on a non-agar substrate. Here, we describe a straightforward coating procedure to construct a chitosan-functionalized microfluidic chamber that fulfills these requirements. This set up circumvents all the disadvantages of traditional agar-based assays, providing new grounds for high-resolution experiments. We also describe simple image processing to maximize the quality of data representation. In theory, our procedure could be used for any bacterial system that adheres to chitosan.
在没有细胞外细胞器的情况下，大量细菌物种能在固体表面平滑移动。在δ-变形菌黄粘球菌中，这种表面运动被称为滑行运动，涉及一种新型大分子机制Agl-Glt。在运动过程中，Agl-Glt系统作为一种完整的包膜蛋白复合物，在细胞腹侧组装。通过这样做，该复合物将表面粘附与Agl运动马达的活性联系起来。在胞质侧，Agl-Glt系统与细菌肌动蛋白细胞骨架MreB相连。有人提出，当表面固定的Agl-Glt复合物在刚性轨道（可能是MreB电缆）上产生牵引力时，就会产生运动。直接验证这一假设既需要微流控技术用抑制剂（即A22、CCCP）干扰运动过程，也需要先进的显微镜技术（即全内反射荧光显微镜和原子力显微镜）。这些方法需要一个显微镜室，细胞在其中在非琼脂底物上的液体中滑行。在这里，我们描述了一种简单的涂层程序，以构建一个满足这些要求的壳聚糖功能化微流控室。这种设置规避了传统琼脂基检测的所有缺点，为高分辨率实验提供了新的基础。我们还描述了简单的图像处理，以最大限度地提高数据表示的质量。理论上，我们的程序可用于任何粘附于壳聚糖的细菌系统。
