Characterization of spores of Bacillus subtilis which lack dipicolinic acid

Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipi colinic acid (DPA) and are too unstable to be purified and studied in detai l. However, the spores of a strain lacking the three major germinant recept ors (termed Delta ger3), as well as spoVF, can be isolated, although they s pontaneously germinate much more readily than Delta ger3 spores. The Delta ger3 spoVF spores lack DPA and have higher levels of core water than Delta ger3 spores, although sporulation ivith DPA restores close to normal levels of DPA and core water to Delta ger3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA, The Delta ger3 spoVF spores also contain minimal levels of the process ed active form (termed P-41) of the germination protease, GPR a finding con sistent with the known requirement for DPA and dehydration for GPR autoproc essing. However, any P-41 formed in Delta ger3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore prot ein (SASP) substrates, SASP-gamma, Analysis of the resistance of wild-type, Delta ger3, and Delta ger3 spoVF spores to various agents led to the follo wing conclusions: (i) DPA and core water content play no role in spore resi stance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core w ater content is associated with decreased spore resistance to wet heat, hyd rogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; ( iii) the absence of DPA increases spore resistance to UV radiation; and (iv ) wild-type spores are more resistant than Delta ger3 spores to Betadine an d glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.