Trends in Microbiology
ReviewI will survive: DNA protection in bacterial spores
Section snippets
Spore DNA resistance
Dormant spores of Bacillus and Clostridium species and their close relatives are formed in sporulation (Box 1; Box 2; Box 3) and can survive for hundreds of years and perhaps longer 1, 2, 3. The physiological properties of these spores are different from those of other known cells – Bacillus and Clostridium spores in water exhibit no detectable metabolism of endogenous or exogenous compounds and can survive treatments with wet and dry heat, UV and γ-radiation, desiccation and toxic chemicals
DNA protection in spores
The spore DNA is located in the core (Box 2), a region that has a lower water content (25–50% wet weight) than the protoplast of a growing cell or the outer spore layers (∼80% of wet weight is water in both) [13]. The spore core also has a pH of ∼6.5, which is ∼1 pH unit lower than in growing cells, and has a huge amount of pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA); ∼20% of core dry weight] 13, 14 (Box 1, Box 2). DPA in spores is almost certainly present as a 1:1 chelate with
α/β-type SASP
The α/β-type SASP are encoded by monocistronic genes, termed ssp, which are members of a multigene family of 3–7 members in spore-formers (Figure 1; Figure 2). The ssp genes are scattered on chromosomes (and on a plasmid in Bacillus cereus ATCC 10987; Figure 1), their promoters are upstream of and close to a strong ribosome-binding site, and coding sequences are followed by potential stem-loop structures that are probably transcription stop signals. Proteins encoded by ssp genes are not
Effects of α/β-type SASP on DNA properties in spores
Studies in vivo and in vitro have shown that α/β-type SASP have profound effects on DNA properties. Studies in vivo have largely used wild-type and α−β−B. subtilis spores. The α−β− spores are significantly more sensitive than wild-type spores to wet and dry heat, UV radiation, dessication and genotoxic chemicals including nitrous acid, hydrogen peroxide and formaldehyde, although α−β− and wild-type spores exhibit similar resistance to γ-radiation and DNA alkylating agents 4, 5, 9, 10, 24 (Table
Effects of α/β-type SASP on DNA in vitro and vice versa
Most of the effects of α/β-type SASP on DNA in spores are also seen in vitro using purified DNA and proteins, including α/β-type SASP from Bacillus and Clostridium species and proteins that are normally abundant and less abundant 4, 6, 7. All wild-type α/β-type SASP have similar effects on DNA in vitro, including slowing DNA depurination due to wet or dry heat, protecting DNA against cleavage by enzymes, hydroxyl radicals and hydrogen peroxide, preventing cytosine deamination to uracil and
Features of α/β-type SASP-DNA binding and the structure of the complex
All α/β-type SASP bind better to GC-rich DNAs, in particular to polydG–polydC, with polydA–polydT bound poorly if at all 14, 15, 34, 35, 39. dG6–dC6 is bound but binding is generally stronger to larger DNAs, especially for oligodATs. Mixed sequence DNAs such as plasmids can be saturated with α/β-type SASP, with apparent affinity constants as high as 107 M−1, although this value varies between different proteins 34, 35, 39. The site size for α/β-type SASP on DNA is 4–5 bp, binding is similar
Interaction of other components with α/β-type SASP-bound DNA in spores
Although the α/β-type SASP alone have striking effects on DNA properties in vitro, the situation in spores is likely to be more complex because additional components interact with DNA. These include the essential HBsu protein found at high levels on DNA in growing cells and spores [44], and Ca-DPA. HBsu decreases the persistence length of DNA saturated with α/β-type SASP in vitro without altering effects of α/β-type SASP on DNA photochemistry [44]. Perhaps the effect of HBsu on DNA persistence
Degradation of α/β-type SASP in spore outgrowth
Although α/β-type SASP are essential to spore DNA resistance, these proteins must be removed to enable DNA transcription in spore outgrowth. Two factors are important in protein removal: (i) dissociation of α/β-type SASP from DNA in fully germinated spores; and (ii) degradation of the proteins initiated by an endoprotease, Gpr, which is specific for α/β-type (and γ-type) SASP [45]. Peptidases then degrade the Gpr cleavage products to amino acids that support much protein synthesis and energy
Concluding remarks and future questions
The α/β-type SASP are the major factor protecting spore DNA from many damaging agents. These proteins are unique and, given their role in protecting DNA in a dormant organism where an entire chromosome is transcriptionally silent, this uniqueness might not be surprising. Indeed, the presence of these proteins in growing cells is extremely deleterious. Although much has been learned about these proteins and their interaction with DNA, several questions remain. These include the following: (i)
Acknowledgements
I thank all past and current members of the laboratory who have contributed to our understanding of the many aspects and effects of α/β-type SASP, Ca-DPA and Gpr, and to collaborators at other institutions, in particular Mark J. Jedrzejas and his coworkers. Work in my laboratory on these topics has been supported by grants from the National Institutes of Health (GM 19698) and the Army Research Office.
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