![]() radiodurans are joined by persistent Holliday junctions. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. Thus, the radiation survival threshold of polyploid D. Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae) and vegetative cells and endospores of three Bacillus bacteria (B. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. The latter finding further suggests that targeting PGM activity during germination could be a novel way to minimize the damaging effects of spores. These findings indicate why 3PGA accumulation during sporulation (and utilization during germination) in all the Firmicute spores studied can be crucial for spore revival due to the generation of essential ATP. However, the 3PGA-replete spores that germinated in the poor medium accumulated >30 times more ATP, and >70% of the germinated spores were found to be alive. The ATP production in the germination of 3PGA-less spores in a poor medium was minimal, and the germinated spores were >99% dead. The current work found no 3PGA in those Bacillus subtilis spores that do not accumulate CaDPA during sporulation and have a core pH of ~7.4. However, in wild-type spore germination, increases in core pH to 7.5–8 and in core water content upon CaDPA release and cortex peptidoglycan hydrolysis allow for rapid 3PGA catabolism, generating ATP indeed, the earliest ATP generated following germination is from 3PGA catabolism. Spores’ 3PGA is stable for months at 4 ☌ and weeks at 37 ☌. This core acidification inhibits phosphoglycerate mutase (PGM) at pH 6.4, allowing 3PGA accumulation, although PGM is active at pH 7.4. The development of Bacillus spore cores involves the accumulation of 3-phosphoglycerate (3PGA) during sporulation, following core acidification to ~6.4, and before decreases in core water content occur due to Ca-dipicolinc acid (CaDPA) uptake. NMR peaks corresponding to ADP or ATP were not detected in these samples, indicating that levels of these two nucleotides were not more than 5% of AMP levels in spores incubated for 10 or 30 days. All values are averages based on duplicate determinations in one experiment, and individual values for 3PGA, AMP, and CMP plus UMP did not deviate by more than 11, 14, and 23%, respectively, from average values. At various times spores were harvested, small molecules were extracted, and levels of 3PGA (A) or AMP (B, filled symbols) or CMP plus UMP (B, open symbols) were determined by 31 P NMR, as described in Materials and Methods. ![]() megaterium GR-less strain GC614 were prepared in a fermentor, isolated, and incubated in the spent sporulation medium plus 200 mM Tris-HCl buffer (pH 8.8) and 20 mM (NH 4 ) 2 SO 4 at either 37☌ (circles) or 50☌ (triangles), giving a pH of 8.6 in the spent medium plus spores, in contrast to a pH of 7 in spent sporulation medium by itself. megaterium spores incubated in spent sporulation medium and with spore core pH adjusted to 7.8. Levels of 3PGA and AMP or CMP plus UMP in dormant B. ![]()
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