Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress

Abstract

Bacterial spores of the genera Bacillus and Clostridium are extremely resistant against desiccation, heat and radiation and involved in the spread and pathogenicity of health relevant species such as Bacillus anthracis (anthrax) or Clostridium botulinum. While the resistance of spores is very well documented, underlying mechanisms are not fully understood. In this study we show, by cryo-electron microscopy of vitreous sections and particular resin thin section electron microscopy, that dormant Bacillus spores possess highly ordered crystalline core structures, which contain the DNA, but only if small acid soluble proteins (SASPs) are present. We found those core structures in spores of all Bacillus species investigated, including spores of anthrax. Similar core structures were detected in Geobacillus and Clostridium species which suggest that highly ordered, at least partially crystalline core regions represent a general feature of bacterial endospores. The crystalline core structures disintegrate in a period during spore germination, when resistance against most stresses is lost. Our results suggest that the DNA is tightly packed into a crystalline nucleoid by binding SASPs, which stabilizes DNA fibrils and protects them against modification. Thus, the crystalline nucleoid seems to be the structural and functional correlate for the remarkable stability of the DNA in bacterial endospores.

Variability in DPA and Calcium Content in the Spores of Clostridium Species

“…The best-studied mechanism of spore resistance is that to UV, achieved with small-acid soluble proteins (SASPs), which bind to the spore’s DNA and promote its conformational change (Setlow, 1995; Raju et al, 2006; Lee et al, 2008). Recently, it was determined that DNA was packed into crystalline nucleoids through binding by α/β type SASPs, hence protecting DNA from modification (Dittmann et al, 2015). Such protein-DNA structures allow spores to endure multiple types of environmental insults such as non-ionizing radiation, wet and dry heat, and desiccation, probably because the movement of internal molecules is restricted (Dittmann et al, 2015).…”

“…Recently, it was determined that DNA was packed into crystalline nucleoids through binding by α/β type SASPs, hence protecting DNA from modification (Dittmann et al, 2015). Such protein-DNA structures allow spores to endure multiple types of environmental insults such as non-ionizing radiation, wet and dry heat, and desiccation, probably because the movement of internal molecules is restricted (Dittmann et al, 2015). In B. subtilis , superdormant spores were identified and were defined as spores able to withstand multiple rounds of germination conditions in the dormant state (Ghosh and Setlow, 2009; Ghosh et al, 2009).…”

Self‐Pressurized Rapid Freezing as Cryo‐Fixation Method for Electron Microscopy and Cryopreservation of Living Cells

“…Since commercial HPF devices are very costly and cannot be easily used in special environments, such as biological safety areas or outside of the laboratory, self‐pressurized rapid freezing (SPRF) has been recently established as a less costly and easy‐to‐use alternative for electron microscopy cryo‐fixation (Han et al., ; Leunissen & Yi, ). As the newest of the HPF procedures, SPRF has been successfully applied for cryo‐fixation in recent years resulting in superb ultrastructure preservation of various samples such as prokaryotes, yeast, plant, and animal cells (Dittmann, Han, Grabenbauer, & Laue, ; Han et al., ; Leunissen & Yi, ; Sarkar et al., ; Yakovlev & Downing, ) (see Figure ).…”

“…Excellent morphology and ultrastructure preservation of prokaryotes, unicellular eukaryotes, and C. elegans was shown after SPRF cryo‐fixation combined with freeze substitution (Grabenbauer et al., ; Han et al., ; Leunissen & Yi, ) (see Figure ). Recently, several mammalian cell types, prokaryotes, yeast S. cerevisiae , and plant A. thaliana cells were successfully cryo‐immobilized by SPRF and analyzed at high resolution by cryo‐electron microscopy after vitreous sectioning (CEMOVIS) (Dittmann et al., ; Han et al., ; Sarkar et al., ). The ultrastructural preservation was in all cases indistinguishable from those obtained after HPF using commercial HPF apparatus (see Figure ).…”

Cellular structural biology as revealed by cryo-electron tomography

“…In this method, frozen hydrated cells or tissues of interest are sectioned with a diamond knife resulting in samples with a thickness of 40-100 μm (Al-Amoudi et al, 2004). Samples that have been studied by CEMOVIS thus far include mitochondria (Hsieh et al, 2006), human skin (Al-Amoudi et al, 2007;Al-Amoudi and Frangakis, 2008), microtubules (Bouchet-Marquis et al, 2007), bacteria (Delgado et al, 2013;Zuber et al, 2008) and bacterial spores (Couture-Tosi et al, 2010;Dittmann et al, 2015). However, this technique has its limitations, mainly due to compression of the thin sections in the direction of cutting, which can complicate the proper interpretation of the data (Al-Amoudi et al, 2005).…”