Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl₃ to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability.United States. Army Research Office (W911F-09-1-0286)United States. Army Research Office (W911NF-09-0001

Dipicolinic Acid Greatly Enhances Production of Spore Photoproduct in Bacterial Spores upon UV Irradiation

Formation of the spore photoproduct (SP) (5-thyminyl-5,6-dihydrothymine) in DNA of dormant spores of Bacillus subtilis upon UV irradiation is due to binding of α/β-type small, acid-soluble proteins (SASP). However, the yield of SP as a function of UV fluence is ∼15-fold higher in spores than in an α/β-type-SASP-DNA complex in vitro. The yield of SP as a function of UV fluence in forespore DNA from mutants which make α/β-type SASP but not dipicolinic acid (DPA) was 10 to 20 times lower than that in dormant spores. Furthermore, the yield of SP as a function of UV fluence in an α/β-type-SASP-DNA complex in vitro was increased sixfold by DPA. These data provide further support for the idea that the high DPA level in dormant spores increases the yield of SP as a function of UV fluence and thereby sensitizes spores to UV. </jats:p

Heat Killing of <i>Bacillus subtilis</i> Spores in Water Is Not Due to Oxidative Damage

ABSTRACT The heat resistance of wild-type spores of Bacillus subtilis or spores (termed α − β − ) lacking DNA protective α/β-type small, acid-soluble spore proteins was not altered by anaerobiosis or high concentrations of the free radical scavenging agents ethanethiol and ethanedithiol. Heat-killed wild-type and α − β − spores exhibited no increase in either protein carbonyl content or oxidized bases in DNA. These data strongly suggest that oxidative damage to spore macromolecules does not contribute significantly to spore killing by heat. </jats:p

Absence of Transient Elevated UV Resistance during Germination of Bacillus subtilis Spores Lacking Small, Acid-Soluble Spore Proteins α and β

Germinating spores of Bacillus subtilis mutants which lack small, acid-soluble spore proteins α and β did not exhibit the transient elevated UV resistance seen during germination of wild-type spores