Effects of Mn and Fe Levels on Bacillus subtilis Spore Resistance and Effects of Mn ²⁺ , Other Divalent Cations, Orthophosphate, and Dipicolinic Acid on Protein Resistance to Ionizing Radiation

Abstract

Spores of Bacillus subtilis strains with (wild type) or without (␣ ؊ ␤ ؊ ) most DNA-binding ␣/␤-type small, acid-soluble proteins (SASP) were prepared in medium with additional MnCl 2 concentrations of 0.3 M 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, ␣ ؊ ␤ ؊ 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 ␣ ؊ ␤ ؊ 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.

Escherichia coli Genes and Pathways Involved in Surviving Extreme Exposure to Ionizing Radiation

“…The requirements for genes involved in protein stability and turnover, as well as those involved in oxidative stress signaling, can likely be understood in the context of current research from the Daly and Radman groups that indicates that protein oxidation is a major deleterious effect of IR (21,24,44,79,80). As is the case for DNA, proteins are a target of IR-mediated damage.…”

Regulation of Manganese Antioxidants by Nutrient Sensing Pathways in Saccharomyces cerevisiae

“…High levels of Mn have also been shown to substitute for SOD in strains of Escherichia coli (Al-Maghrebi et al 2002), Neisseria gonorrheae (Tseng et al 2001), and the yeast Saccharomyces cerevisiae (Chang and Kosman 1989;Sanchez et al 2005;Reddi et al 2009), engineered to lack SOD enzymes. Elegant work by Daly and colleagues has shown that tolerance to radiation and oxidative stress in a variety of bacterial species is due to accumulation of high levels of intracellular Mn, but low Fe (Daly 2006(Daly , 2009Daly et al 2007Daly et al , 2010Gross 2007;Granger et al 2011). In higher organisms, Mn treatment can prolong the life span and oxidative stress resistance in the simple metazoan, Caenorhabditis elegans, and also defend against reactive oxygen species (ROS) in the cryopreservation of sperm (Lin et al 2006;Bansal and Kaur 2009;Cheema et al 2009).…”

HtrC Is Involved in Proteolysis of YpeB during Germination of Bacillus anthracis and Bacillus subtilis Spores

“…While most of the Ca 2ϩ released during germination is likely bound to DPA, and our findings indicate that the Ca 2ϩ -DPA complex is ineffective in promoting HtrC activity, it is possible that enough free Ca 2ϩ is present to increase HtrC activity during germination. Spores also contain significant amounts of Mn 2ϩ (40,41), which is likely complexed with DPA and released during germination and could thus play a role in stimulating HtrC.…”