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

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.

This project represents the next stage towards our aim of a complete visualization of the 3D structure and assembly of the spore of the Bacillus cereus family in molecular detail. The project will involve the complementary use of high resolution electron crystallographic techniques combined with mutagenesis. We will build on our previous preliminary characterization of several two-dimensional crystalline layers within the spore. This was mainly restricted to the use of negative stain to reveal the low resolution architecture of the putative exosporium basal and parasporal layers and putative pitted layer of the spore coat. We did however demonstrate that the parasporal layer was amenable to high resolution cryomicroscopy (better than 0.7 nm). We will now aim to apply similar techniques to the basal layer, extend the resolution for the parasporal layer towards atomic detail and extend the study of the pitted layer in to three dimensions. We will determine the protein composition of these different layers, with initial emphasis on the basal layer. We will combine the structural work with genetic and biochemical analysis to define the proteins making up these crystalline layers and start to explore the composition of the space between the layers. We will construct mutants lacking particular exosporium proteins and examine their gross exosporium structure. We will determine 3D difference maps in molecular detail, between wild-type and selected mutant assemblies of these layers, in order to start mapping the location of individual protein components within the structure. We will start with the ExsF and BclA mutants already available. We will determine the function of the exosporium-associated BA1021 protein, encoded in a wide range of exosporium-forming species, including Clostridia. Finally, we will start to explore the protein complement of the sub-exosporial compartment of B. cereus spores, and begin to determine their function by knockout mutation.