ACTIVATORS AND INHIBITORS OF GERMINATION 71 



metal activator required for the enzymatic reactions leading to germination. 

 Experiments were conducted to test this hypothesis. An analysis of the 

 metal change during spore germination with EDTA was made with an 

 emission spectrograph and the information from this analysis was used to 

 study the process in more detail. It was found that copper, iron, magnesium, 

 manganese and calcium were removed by the EDTA. 



The affinity constants of the various metals with EDTA were used to pre- 

 pare EDTA metal complexes in an attempt to determine the metal inhibitor 

 that was being removed during the germination process. If the prepared 

 metal-EDTA complex was lower in the series (affinity constant), theoretically 

 there would be an exchange reaction taking place and germination would 

 occur. The results from experiments using this technique indicated that the 

 metal inhibitor was probably a member of the "light" group of elements. 



A study of environmental conditions affecting the germination process by 

 EDTA indicated a very rapid reaction that could occur between pH of 5.0 

 and 9.4. The germination process by EDTA was almost completely stopped 

 at 0°C. It is probable that at this temperature the chelating ability of the 

 EDTA was not markedly reduced but the subsequent enzymatic reactions 

 associated with germination were adversely affected by the decrease in tem- 

 perature. The effect of inhibitors on spore germination by EDTA indicated 

 that sodium azide, 2,-4,-dinitrophenol and iodoacetate had essentially no 

 effect on the process. 



A study of the metal inhibition suggested that it could be the beryllium 

 inhibition of an alkaline phosphatase. It is logical that phosphatase activity 

 could take place as one of the initial reactions because it is an energy yielding 

 mechanism. Efforts to detect beryllium in or on the spore indicated that it 

 was not present to the extent of 1 ppm or greater. Beryllium was able to 

 block the germination process by EDTA when added at a concentration of 

 6.5 X 10-=^ M. 



Treatment of the spores with excess EDTA followed by washing to remove 

 the EDTA and adding back various metals in an attempt to cause the germina- 

 tion process to proceed were essentially negative. However, magnesium gave 

 slightly positive results. The failure to germinate such spores by the addi- 

 tion of various metal ions was probably due to the disturbance of a metal 

 complex, the disruption of which was irreversible. 



Phosphatase activity was demonstrated in a spore extract obtained by 

 crushing the spores with glass beads. A rate of reaction was calculated for 

 the phosphatase enzyme when pyrophosphate was used as the substrate. The 

 rate of splitting was 19 /xg of P/mg dry wt. of spores/hr. The phosphatase 

 reaction was stopped by the addition of beryllium sulfate. 



