THE FORMATION OF SPORES BY BACTERIA 125 



ments that the interference with sporulation in this case is due not to a 

 drop in the pH but rather to a specific efiFect of ethyl malonate. 



Figure 9 shows the efi^ect produced with diethyl succinate as an 

 inhibitor. Here again the inhibition occurs whether the inhibitor is 

 added before or after the pH begins to rise. Here also, as in the case 

 of the ethyl malonate, the pH rises for awhile and then drops. Figure 

 10 shows the effect produced with ethyl pyruvate. Here also, the in- 

 hibitor functions whether it is added before or after the pH begins to 

 rise, indicating that this inhibitor, as well as the other two, probably 

 interferes with the functioning of some enzyme system rather than 

 with the production of an adaptive enzyme. The ethyl pyruvate acts 

 somewhat differently from the two inhibitors cited above, because in 

 this case the pH rises and stays high. Nevertheless, no spores are 

 formed. We have also investigated the effect of various organic acids 

 upon the reversal of inhibition of these ethyl esters. I am not going to 

 take time to discuss the details of all these experiments; suffice it to say 

 that these inhibitors were reversed by all of the intermediates in the 

 glyoxylic-acid shunt but were not reversed by fumarate or other inter- 

 mediates in the TCA cycle not common- to the glyoxylic-acid shunt. 



We realize it is dangerous to rely upon inhibitors alone for the 

 verification of a definite pathway in a fermentation, but the circumstan- 

 tial evidence we had for the involvement of the glyoxylic-acid shunt led 

 us to conduct further experiments to see if we could get additional sup- 

 port for this conclusion. We therefore investigated two other possible 



8 



pH 6 



3x 10 8 



^^--^ <I0 4 



Ethyl pyruvate 



Addition of ester 



4 8 12 



TIME, HOURS 



Figure 10. The effect of ethyl pyruvate (1.5 X lO'^ M) on the pH and sporulation of 

 Bacillus cereus T. 



