Spores of Bacillus subtilis strains with (wild type) or without (α(-)β(-)) most DNA-binding α/β-type small, acid-soluble proteins (SASP) were prepared in medium with additional MnCl(2) concentrations of 0.3 μM to 1 mM. These haploid spores had Mn levels that varied up to 180-fold and Mn/Fe ratios that varied up to 300-fold. However, the resistance of these spores to desiccation, wet heat, dry heat, and in particular ionizing radiation was unaffected by their level of Mn or their Mn/Fe ratio; this was also the case for wild-type spore resistance to hydrogen peroxide (H(2)O(2)). However, α(-)β(-) spores were more sensitive to H(2)O(2) when they had high Mn levels and a high Mn/Fe ratio. These results suggest that Mn levels alone are not essential for wild-type bacterial spores' extreme resistance properties, in particular ionizing radiation, although high Mn levels sensitize α(-)β(-) spores to H(2)O(2), probably by repressing expression of the auxiliary DNA-protective protein MrgA. Notably, Mn(2+) complexed with the abundant spore molecule dipicolinic acid (DPA) with or without inorganic phosphate was very effective at protecting a restriction enzyme against ionizing radiation in vitro, and Ca(2+) complexed with DPA and phosphate was also very effective in this regard. These latter data suggest that protein protection in spores against treatments such as ionizing radiation that generate reactive oxygen species may be due in part to the spores' high levels of DPA conjugated to divalent metal ions, predominantly Ca(2+), much like high levels of Mn(2+) complexed with small molecules protect the bacterium Deinococcus radiodurans against ionizing radiation.