Characterization of Spores of Bacillus subtilis Which Lack Dipicolinic Acid

Abstract

Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Δ ger3 ), as well as spoVF , can be isolated, although they spontaneously germinate much more readily than Δ ger3 spores. The Δ ger3 spoVF spores lack DPA and have higher levels of core water than Δ ger3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to Δ 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 Δ ger3 spoVF spores also contain minimal levels of the processed active form (termed P 41 ) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P 41 formed in Δ ger3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-γ. Analysis of the resistance of wild-type, Δ ger3 , and Δ ger3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen 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 Δ ger3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.