Architecture of the exosporium and spore coat layers of the Bacillus cereus family

许多致病细菌，包括炭疽病的病原体和食物中毒的病原体，都能够以休眠孢子的形式在恶劣的环境条件下生存。当条件变得更有利时，这些孢子可以萌发，从而允许细菌细胞生长和繁殖。孢子可以在新陈代谢不活跃的状态下存活多年，直到条件变得有利于萌发。孢子能抵抗极端温度、辐射、干燥、刺激性化学物质和物理损害。孢子有一个包含细菌遗传物质的致密核心；它被许多层蛋白质、碳水化合物和脂肪包围，这些层排列得很像洋葱的层。最外层，也就是所谓的“外孢子体”，其作用就像一层薄薄的半多孔膜，上面有一排密集的丝状物，以“毛状绒毛”的形式连接在一起。这一外层很重要，因为它是孢子和环境之间的第一个接触点，例如在被宿主细胞吞噬的炭疽孢子中；它允许孢子附着在表面；它是免疫系统识别的孢子的主要部分；它可能对孢子起到保护作用。我们试图了解孢子的构造方式，以及这种构造是如何赋予孢子特殊的生物学和物理特性的。为了充分了解孢子的工作原理，我们需要了解其分子细节的三维结构。我们还需要知道分子构件是什么，以及它们是如何组合在一起的。我们建议使用电子显微镜来显示不同层的结构，从外孢子体开始，一直向内延伸到孢子核。我们的优势是这些层中的许多层可以分离，使我们能够分析生化成分。此外，它们中的一些是部分结晶的，这使得分子比其他情况下更容易可视化。要建立孢子的三维图像，重要的一步是确定各种类型的蛋白质构建块的位置。我们将通过对细菌进行基因改造来制造缺失已知蛋白质的孢子，然后我们将看到这些蛋白质在三维结构中丢失的位置。这也将告诉我们这些特定的蛋白质以何种方式对孢子的功能和特性起着重要作用。

A number of pathogenic bacteria including the causative agent of anthrax and agents of food poisoning are able to survive harsh environmental conditions in the form of dormant spores. When conditions become more favourable, these spores can germinate and so allow bacterial cells to grow and multiply. Spores can survive in a metabolically inactive state for many years until conditions become favourable for germination. Spores are resistant to extreme temperatures, radiation, desiccation, harsh chemicals, and physical damage. The spore has a dense core containing the bacterium's genetic material; this is surrounded by many layers of protein, carbohydrate and fatty lipids arranged much like the layers of an onion. The outermost layer, know as the 'exosporium' acts like a thin, semi-porous membrane with a dense array of filaments attached in the form of a 'hairy nap'. This outer layer is important because it acts as the first point of contact between the spore and its environment, for example in an anthrax spore engulfed by a host cell; it allows spores to stick to surfaces; it is the main part of the spore recognised by the immune system; and it may have a protective role for the spore. We are attempting to understand the way the spore is constructed and how this construction endows the spore with its special biological and physical properties. In order to fully understand how the spore works we need to understand its three-dimensional structure in molecular detail. We also need to know what the molecular building blocks are and how they are put together. We propose to use an electron microscope to visualise the structure of the different layers, starting with the exosporium and working our way inwards towards the spore core. We have the advantage that many of these layers can be separated, allowing us to analyse the biochemical composition. Moreover, a number of them are partially crystalline making the molecules very much easier to visualise than would otherwise be the case. An important step in building up a three-dimensional picture of the spore will be to identify the positions of the various types of protein building block. This we will do by genetically modifying the bacterium to make spores that are missing known proteins and we will see where these are lost within the three-dimensional structure. This will also tell us in what way these particular proteins are important for the function and properties of the spore.