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In pathogenic Bacillota, spores can form an infectious particle and can take up a central role in the environmental persistence and dissemination of disease. A poorly understood aspect of spore-mediated infection is the fibrous structures or 'endospore appendages' (ENAs) that have been seen to decorate the spores of pathogenic Bacilli and Clostridia. Current methodological approaches are opening a window on these long enigmatic structures. Using cryoID, Alphafold modelling and genetic approaches we identify a sub-class of robust ENAs in a Bacillus paranthracis foodborne outbreak strain. We demonstrate that L-ENA are encoded by a rare three-gene cluster (ena3) that contains all components for the self-assembly of ladder-like protein nanofibers of stacked heptameric rings, their anchoring to the exosporium, and their termination in a trimeric 'ruffle' made of a complement C1Q-like BclA paralogue. The role of ENA fibers in spore-spore interaction and the distribution of L-ENA operon as mobile genetic elements in B. cereus s.l. strains suggest that L-ENA fibers may increase the survival, spread and virulence of these strains., (© 2024. The Author(s).)

Scanning electron microscopy (SEM) can reveal the ultrastructure of bacterial spores, including morphology, surface features, texture, spore damage, germination, and appendages. Understanding these features can provide a basis for adherence, how physical and environmental stressors affect spore viability, integrity, and functionality, as well as the distribution and function of surface appendages. However, the spore sample preparation method can significantly impact the SEM images' appearance, resolution, and overall quality. In this study, we compare different spore preparation methods to identify optimal approaches for preparation time, spore appearance and resolved features, including the exosporium and spore pili, for SEM imaging. We use Bacillus paranthracis as model species and evaluate the efficacy of preparation protocols using different fixation and drying methods, as well as imaging under room- and cryogenic temperatures. We compare and assess method complexity to the visibility of the spore exosporium and spore appendages across different methods. Additionally, we use Haralick texture features to quantify the differences in spore surface appearance and determine the most suitable method for preserving spore structures and surface features during SEM evaluation. The findings from this study will help establish protocols for preparing bacterial spores for SEM and facilitating accurate and reliable analysis of spores' characteristics., Competing Interests: Conflict of Interest The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America.)