Characterization of Spores of <i>Bacillus subtilis</i> Which Lack Dipicolinic Acid

TLDR

Spores of Bacillus subtilis lacking spoVF cannot synthesize dipicolinic acid, rendering them unstable and difficult to purify. Δ ger3 spoVF spores lacking DPA can be isolated but germinate more readily, have higher core water, minimal processed GPR, and reveal that DPA and core water do not influence resistance to dry heat, desiccation, or glutaraldehyde, whereas elevated core water reduces resistance to wet heat, hydrogen peroxide, formaldehyde, and Betadine, and DPA absence increases UV resistance.

Abstract

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.