Germination of Bacterial Endospores With Subtilopeptidases

Abstract

Intact spores of Bacillus subtilis are susceptible to subtilopeptidase attack and this enzymatic reaction induces changes in the spore similar to those that take place during "physiological" germination. Germination occurred between pH 5.5 and 10.0 showing a pH optimum of 9.0 and between 25 °C and 45 °C with an optimum of 37–40°. Subtilopeptidase-induced germination took place in completely anaerobic conditions. Sublethal heating of spore suspensions increased the rate of subtilopeptidase-induced germination. Germination with subtilopeptidase was almost completely inhibited by an excess of diisopropyl fluorophosphate. L-Alanine-induced germination was not affected by diisopropyl fluorophosphate. Participation of the spore metabolism in subtilopeptidase-induced germination seemed likely. These results suggest that subtilopeptidases initiate spore germination by releasing germination agents from the spore.No significant loss of viability was noted until after exposure to the proteolytic enzyme for at least 60 minutes. Prolonged exposure of B. subtilis spores to subtilopeptidase results in death of the exposed spores.Subtilopeptidase-induced germination was also observed in several spores of other members of the genus Bacillus.

(Symposium on Bacterial Spores : Paper IV). Germination and the Problem of Dormancy

“…This protease, produced by B. subtilis, will cause germination of some spores (Sierra, 1967). Inhibition by malanine, synergism with inosine for germination of B. cereus spores and diffusibility of the germinative molecule suggested that subtilisin acted by autodigestion to form traces of germinative L-alanine (Gould & King, 1969).…”

“…Inhibition by malanine, synergism with inosine for germination of B. cereus spores and diffusibility of the germinative molecule suggested that subtilisin acted by autodigestion to form traces of germinative L-alanine (Gould & King, 1969). Nevertheless, subtilisin is one of the few proteases which will partly degrade isolated spore coats (Sierra, 1967;Gould & King, 1969), from which traces of L-alanine may be released.…”

Mechanism and Factors Influencing the Veronal Inhibition of Bacterial Spore Germination

“…niger strain globigii (Microbiological Research Establishment, Porton, England) was also used in this study. The organisms were grown in a medium containing Nutrient Agar supplemented with MnSO4 1H20 and NaCl to essentially complete and synchronous sporulation as previously described (8). After keeping the cultures for 3 days at 4 C, the spores were harvested and washed extensively with deionized water.…”

“…Consequently, as a rule, before use in each experiment, the spores were washed, uniformly resuspended, and diluted in 0.8 mm Tween 80-deionized water. The standardization of the spore suspensions and the determination of the spore concentration of the suspensions were accomplished as described previously (8). The percentage of reduction in absorbancy was a reliable indication of the percentage of spores which completed the initiation phase.…”

Sporulation and germination of spores of Clostridium perfringens

“…Rode and Foster (1962a) suggested that ions could affect some structure protective to the core which depended on associated ions for its stability, the most likely candidate for this role being the electronegative cortex murein. Rode and Foster (1965) (Sierra, 1964(Sierra, , 1967Gould and Hitchins, 1965) which are thought to* be involved in germination. Slepecky (1963)…”