title: Ellipsoid Localization Microscopy Infers the Size and Order of Protein Layers in Bacillus Spore Coats.
abstract: Multilayered protein coats are crucial to the dormancy, robustness, and germination of bacterial spores. In Bacillus subtilis spores, the coat contains over 70 distinct proteins. Identifying which proteins reside in each layer may provide insight into their distinct functions. We present image analysis methods that determine the order and geometry of concentric protein layers by fitting a model description for a spheroidal fluorescent shell image to optical micrographs of spores incorporating fluorescent fusion proteins. The radius of a spherical protein shell can be determined with <10 nm error by fitting an equation to widefield fluorescence micrographs. Ellipsoidal shell axes can be fitted with comparable precision. The layer orders inferred for B. subtilis and B. megaterium are consistent with measurements in the literature. The aspect ratio of elongated spores and the tendency of some proteins to localize near their poles can be quantified, enabling measurement of structural anisotropy.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Julia
authors.initials: J
authors.lastname: Manetsberger
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: James D
authors.initials: JD
authors.lastname: Manton
authors.affiliation: Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary.
authors.forename: Miklos J
authors.initials: MJ
authors.lastname: Erdelyi
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Henry
authors.initials: H
authors.lastname: Lin
authors.affiliation: Malvern, Worcestershire, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Rees
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Graham
authors.initials: G
authors.lastname: Christie
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom. Electronic address: ejr36@cam.ac.uk.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rees
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D013171:Spores, Bacterial / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D001426:Bacterial Proteins; C030069:spore-specific proteins, Bacillus