Pushing the envelope: atomic force microscopy imaging of the bacterial outer membrane during growth and division

革兰氏阴性菌是最有可能对常用抗生素产生耐药性的微生物之一，因此，以前无害的感染可能变成严重的、可能危及生命的疾病。这在一定程度上是因为这些细菌（包括鲍曼不动杆菌、铜绿假单胞菌和沙门氏菌和大肠杆菌等肠杆菌科细菌）受到外膜的保护，该外膜限制了抗生素进入细胞。基于光学显微镜，特别是荧光显微镜，有人认为这种外膜以相当复杂的方式组织其组成部分，包括形成蛋白质岛，这可能也在细菌清除抗生素的效率中发挥作用。一般来说，光学显微镜是发现和理解定义细胞的生命、生长、分裂和死亡的生物现象的最有力的工具。然而，它对活细胞的分辨率大多不足以在分子长度尺度上解析细胞。原子力显微镜（AFM）是一种替代技术，它可以通过用锋利的探针轻轻追踪单个分子的轮廓来探测它们。我们最近使用原子力显微镜在分子尺度上解析活细菌的外膜，从而揭示了膜是如何分离成不同的富含蛋白质和富含脂质的结构域的。在这里，我们注意到细菌在有利的条件下每20分钟就可以分裂一次，我们建议开发方法，使我们能够对生长和分裂的细胞进行这种AFM实验，目的是更好地了解外膜如何促进/适应细菌的生长和分裂，包括合成和插入新的膜成分。

Gram-negative bacteria are among the microbes with the highest potential to develop resistance against commonly used antibiotics, such that previously harmless infections can become severe and potentially life-threatening diseases. This is in part because these bacteria (including A. baumannii, P. aeruginosa, and enterobacteriaceae such as Salmonella and E. coli) are protected by an outer membrane that limits entry of antibiotics into the cell.Based on light - and particularly fluorescence - microscopy, it has been suggested that this outer membrane organises its building blocks in rather sophisticated ways, including the formation of protein islands that may also play a role in how efficiently bacteria clear antibiotics. Quite generally, light microscopy has been a most powerful tool to discover and understand biological phenomena that define the life, growth, division, and death of cells. Yet its resolution on living cells is mostly insufficient to resolve cells at molecular length scales.Atomic force microscopy (AFM) is an alternative technique that can probe single molecules by gently tracing their contours with a sharp probe. We have recently used AFM to resolve the outer membrane of living bacteria at molecular-scale resolution, thereby revealing how the membrane segregates into different protein-enriched and lipid-enriched domains.Here, noting that bacteria can divide every ~20 minutes under favourable conditions, we propose to develop methods that will enable us to carry out such AFM experiments on growing and dividing cells, with the aim to better understand how the outer membrane facilitates/adapts to bacterial growth and division, including the synthesis and insertion of new membrane components.