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.

Structured illumination microscopy combined with machine learning enables the high throughput analysis and classification of virus structure

“…The width of these structures appeared to be limited by the resolution of the imaging technique (~90 nm for TIRF-SIM, see Figure S1 but also observed in higher resolution approaches such as dSTORM) and therefore, they were considered as 1D structures. The large spherical structures were analysed with the recently-developed technique, ellipsoid localization microscopy (ELM) 17 . This approach constitutes a form of model fitting to imaging data, which we showed allow the extraction of structural parameters beyond the image resolution 6,17 .…”

“…The large spherical structures were analysed with the recently-developed technique, ellipsoid localization microscopy (ELM) 17 . This approach constitutes a form of model fitting to imaging data, which we showed allow the extraction of structural parameters beyond the image resolution 6,17 . However, we observed no significant ellipsoidicity and therefore we applied the spherical approach of ELM, extracting the radius of the particles.…”

“…Once classified, these uniform populations can be analysed via image analysis pipelines specifically adapted to each structural class, such as image model fitting. We and others have shown that appropriate model fitting can lead to precision in structural parameters beyond the resolution of the images used 3,17 . This allows an in-depth exploration of virus populations unachievable by electron microscopy (EM) by allowing the measurement of large numbers of particles with high specificity.…”

The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

“…B. subtilis spores are negatively charged (Huang et al, 2010; Pesce et al, 2014) and have a relative hydrophobicity that is in part due to the glycosylation of some spore surface proteins (Cangiano et al, 2014; Rusciano et al, 2014). In several Bacillus and Clostridium species, including B. cereus, B. anthracis, B. megaterium , and C. difficile , the outermost spore structure is the exosporium, a morphologically distinct layer composed of proteins and glycoproteins that surrounds the coat (Díaz-González et al, 2015; Manetsberger et al, 2015b; Stewart, 2015). …”

Proteins Encoded by the gerP Operon Are Localized to the Inner Coat in Bacillus cereus Spores and Are Dependent on GerPA and SafA for Assembly

“…Bioinformatic analyses do not reveal any functional clues, only that the various GerP proteins largely resemble orthologous proteins in other Bacillus species. Hence, the purpose of the current study was to use the recently developed ellipsoid localization microscopy (ELM) technique (9) to more precisely determine the location of the various GerP proteins in Bacillus cereus spores. We sought additionally to ascertain whether there is any dependency between the various GerP proteins, and between the GerP and coat morphogenetic proteins, for localization in the spore.…”