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.

Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye

“…This positive staining is observed at least 1 h before the phase-dark spore formation, which is the microscopic indication of germinating spores. A study showed that 3-4 min after the initiation of germination of B. subtilis 1,604 strain, production of ATP is detected (Venkatasubramanian and Johnstone 1989). Fluorescence monitoring of NADH and dosage of ATP in germinating spore of B. megaterium QM B1551 also support this conclusion (Sano et al 1988).…”

Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis

“…Preparation of autolysin-containing fractions. Unless otherwise stated, autolysin-containing samples were prepared Prior to germination, cleaned, dormant spores at 5 mg/ml (wt/vol) were heat activated at 70°C for 60 min (37). Heatshocked spore suspensions were germinated at 5 mg/ml at 37°C with constant shaking in 1 mM L-alanine-100 p.g of chloramphenicol per ml-50 mM potassium phosphate buffer (pH 7.0).…”

The gerB region of the Bacillus subtilis 168 chromosome encodes a homologue of the gerA spore germination operon

“…Mutations in gerA are the only ones known to specifically block the ALA response without affecting the AGFK response. In contrast a complex set of germinants is required for the AGFK response, the response is inhibited by sodium azide (Venkatasubramanian & Johnstone, 1989) and mutants of gerB and gerK are specifically blocked in the response.…”