Biochemical Analysis of the Bacillus subtilis 1604 Spore Germination Response

Abstract

Germination at 37 degrees C of spores of Bacillus subtilis 1604 in the L-alanine and potassium phosphate (ALA) and the glucose, fructose, L-asparagine, potassium chloride (GFAK) germinant systems was triggered following heat activation at 70 degrees C for 1 h. In these conditions, 50% of the spore population became committed to germinate after exposure for 10 min and 14 min to ALA and GFAK, respectively, at which time 38% and 30% losses of OD600 had taken place. Dipicolinic acid (DPA) release, loss of heat resistance and release of soluble hexosamine-containing fragments occurred after commitment and were closely associated with loss of refractility in both the ALA and GFAK pathways. Net ATP synthesis could not be detected until 3-4 min after initiation of germination in both ALA and GFAK, by which time greater than 20% of the spore population was committed to germinate. The ALA and GFAK germination pathways were greater than 99% inhibited by 3 and 1 mM-HgCl2, respectively, as measured by OD600 loss. Reversible post-commitment HgCl2-sensitive sites were present in the ALA and GFAK pathways which were 50% inhibited by 0.125 mM and 0.05 mM-HgCl2, respectively. A pre-commitment HgCl2-sensitive site was identified in the ALA pathway which was 55% inhibited by 6 mM-HgCl2. At 3 mM-HgCl2, 70% of the spore population became committed to germinate in the ALA pathway, whereas less than 5% OD600 loss occurred. In this system, loss of heat resistance was associated with commitment, whereas OD600 loss and DPA release were identified as post-commitment events. The ALA and GFAK pathways were insensitive to a variety of metabolic inhibitors. Protease inhibitors had different effects on the ALA and GFAK pathways: phenylmethanesulphonyl fluoride (PMSF) solely inhibited ALA germination at a pre-commitment site and had little effect on GFAK germination, whereas N alpha-p-tosyl-L-arginine methyl ester (TAME) inhibited both the ALA and GFAK pathways at pre- and post-commitment sites. These results are discussed in relation to a recently proposed model for the triggering of Bacillus megaterium KM spore germination.