Ultrastructural details of resistance factors in Bacillus subtilis spores revealed by cryo-FIB milling and electron tomography
Résumé
Bacterial spores are dormant cells that can withstand a wide range of stresses, including
antibiotics, detergents, irradiation and high temperatures. Such resilience is an asset when
spores are used for the benefit of humans, as in the case of probiotics or spore-based delivery
technologies. On the other hand, it represents a major problem in terms of infectious diseases,
food safety or biological warfare when it comes to spores of pathogenic bacteria. Despite their
importance for public health and industry, the assembly, architecture and composition of the
molecular structures contributing to spore resistance are not yet fully elucidated. A major
constraint in understanding these features lies in their nanometric dimensions, which require
high-resolution observation methods to be studied.
Cryo-electron tomography (Cryo-ET) meets this need and opens wide frontiers towards the
three-dimensional visualization of ultrastructures and molecules in the cell. The resolution
power of cryo-ET is however limited by the penetration of electrons, thus requiring sample
thinning to reach molecular details. In the past decade, cryo-focused ion beam milling
monitored by scanning electron microscopy (cryo-FIB/SEM), emerged as a state-of-the-art
sectioning technique to generate cell lamellae with nanoscale precision.
In our work, we have implemented cryo-FIB/SEM-ET to access deep cell regions and shed light
on ultrastructures of sporulating Bacillus subtilis cells. Our tomograms show a wide variety of
cellular ultrastructures with unprecedented level of details, including DNA compaction in the
forespore, the architecture of the mother cell and forespore envelopes, and early coat layers.
Segmentation was performed to get a three-dimensional overview of the cellular content. In
parallel, we investigated the nature of the nascent coat layers in various mutant strains using
cryo-FIBM/ET and transmission electron microscopy on resin sections of freeze-substituted
bacteria. Combining these two cellular electron microscopy approaches, we distinguish seven
nascent coat regions with different molecular properties, and propose a model for the
contribution of the morphogenetic proteins SpoIVA, SpoVID, SafA and/or CotE.
Domaines
Sciences du Vivant [q-bio]Origine | Fichiers produits par l'(les) auteur(s) |
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