ENZYMES DORMANT IN THE INTACT SPORE 111 



Means of Activation. Depending upon the species, strain, history, etc., 

 the dormant enzymes of the intact spore may be activated in the absence of 

 any detectable germination by treatment with small amounts of appropriate 

 germinating nutrients, and/or heating. 



Nature of Activation. Inasmuch as the oxidative enzymes as well as other 

 enzymes dormant in the intact spore are active in spore-free extracts, one 

 begins to wonder seriously about the nature of the activation process and 

 the causes for dormancy and heat stability. That considerations of permea- 

 bility alone cannot fully explain the nature of the activation process is evident 

 when one recalls that, in some cases at least, the activity of the extracts was 

 increased by heat shocking the spores prior to rupture. We therefore started 

 looking for other changes during heat activation — particularly the release of 

 dipicolinic acid from the spores. The total dipicolinic acid content of the 

 spores was 6.6-7.0% on a dry weight basis. We have noted a partial release 

 of dipicolinic acid during heating with alanine, and subsequent incubation 

 at room temperature and oxidation of glucose, on germination and on auto- 

 claving (Table VII). Partial release of dipicolinic acid has also been ob- 

 served by Harrell (1956) during the heat activation of aged spores of B. 

 terminalis. On complete germination or autoclaving, most of the dipicolinic 

 acid of the spores is liberated into the medium and, presumably, the total 

 oxidative enzymes associated with growth from spores are completely acti- 

 vated and rendered sensitive to heat, inasmuch as the germinated spores 

 become nonviable even on 15 min. exposure to 65 °C. In marked contrast, 

 no decrease in the viable count of heat-stable spores could be detected by 



Table VII 



Dipicolinic acid content of B. terminalis spores and release of 



dipicolinic acid on germination and activation by heat shock 



in the presence of nutrients 



Time % of dipicolinic acid released 



Immediately after heat shock 



1 hr. after heat shock 



After 30 minutes of oxidation of 



glucose (90 minutes after heat shock) 

 30 minutes after germination 

 On autoclaving 



Strength of spore suspension — 15 mg dry wt/ml 

 JNutrients— 26 fig/m\ L-alanine; 6 /tg/ml adenosine 

 Substrate — Glucose 0.5 mg/ml 

 Duration of heat shock — 5 hrs. at 65 °C 

 Dipicolinic acid content of spores on dry wt — 6.6% 



