Abstract

Multi-layered protein coats are used for environmental protection, sensing, and interaction by many microorganisms, including spore-forming bacteria and viruses. In spores of Bacillus subtilis, over 70 distinct proteins make up a coat that is only about 100 nm thick, which helps keep the spore viable and able to germinate after a time as long as several decades in a harsh environment. Distinguishing the order of protein layers can indicate the function of different proteins – for example, which proteins form the outermost layers that protect the spore from lytic enzymes, and which hold the structure together? While fluorescent fusion proteins provide the only practical and non-invasive way to identify specific proteins in these multi-layered specimens, conventional optical microscopy lacks the resolution to resolve adjacent protein layers. Our original methods of fluorescent shell localisation make it possible to determine the order and geometry of concentric protein layers by fitting mathematical model structures to image data. Our method determines the radius of protein shells to a precision better than 10 nm, and produces layer orders for Bacillus subtilis and megaterium consistent with previous electron mi­croscopy studies. In addition, the aspect ratio of elongated spores and the tendency of some pro­teins to localise near the poles can be quantified, enabling measurement of structural anisotropy. In future work, this technique will be used to optimise the structure of bacterial strains being developed for therapeutic drug delivery, in a joint project with MedImmune.