THE FORMATION OF SPORES BY BACTERIA 127 



inhibitors. If either the TCA or the glyoxylic shunt (the cycles are 

 shown in Figure 11) is involved, fluoroacetic acid should also be an 

 effective inhibitor, inasmuch as this material interferes with the enzyme 

 that converts citrate to isocitrate. We found this acid to be an effective 

 inhibitor of sporulation. It did not interfere with the growth of the 

 vegetative cells. Thus it functions very much like the esters we reported 

 above. This inhibitor was reversed by citrate, isocitrate, succinate, and 

 malonate but not by fumarate, acetate, pyruvate, or alpha-ketoglu- 

 tarate. 



We also tried sodium bisulphite as an inhibitor, reasoning that, if 

 the glyoxylic-acid shunt is involved, bisulphite should tie up the gly- 

 oxylic acid because of its aldehyde group, and thus break the cycle; of 

 course, it may also tie up the ketone group of the oxalacetate, which 

 is common to both the glyoxylic-acid shunt and the TCA cycle. In any 

 event, we found that bisulphite did effectively interfere with sporula- 

 tion but did not interfere with the growth of the vegetative cells. This 

 inhibitor was reversed by citrate, cis-aconitate, isocitrate, succinate, 

 methyl malonate, malonate, and glyoxylate. It was not reversed by 

 pyruvate, acetate, alpha-ketoglutarate,. aspartate, or malate. The re- 

 versal by glyoxylic acid and ketoglutarate was to be expected, since 

 the aldehyde and ketone groups would tie up the bisulphite and thus 

 remove it from the sphere of action. The fact that malate does not re- 

 verse the inhibition of the bisulphite may indicate that bisulphite is 

 also t\'ing up the oxalacetate and thus breaking the cycle at that point. 



The glyoxylic-acid shunt may be needed for sporulation, but as 

 yet we do not have convincing proof. At the present time we are pur- 

 suing this investigation with radioactive tracers, and hope by this tech- 

 nique to get conclusive proof or denial. Regardless of the cycle in- 

 volved, all of the inhibitors we have studied are reversed by succinate, 

 and succinate has proved to be the most effective reversing agent be- 

 cause it will reverse these inhibitors in smaller concentrations than any 

 of the others. This leads us to suspect that succinic acid is an inter- 

 mediate in the synthesis of spore material and also, perhaps, in the 

 synthesis of DPx\. There is some evidence against this conclusion; 

 Martin and Foster ( 1958 ) , when they studied the incorporation of 

 various types of labeled compounds into DPA, obtained little evidence 

 for the incorporation of succinate. In their experiments there may have 

 been an abundant supply of succinate within the cell so that the cell 

 did not utilize succinate added from the outside. You may recall from 

 the data on the anaerobic culture that we could separate the formation 

 of the heat-sensitive spore from the synthesis of DPA and from the 

 development of heat resistance. We have not been able to obtain such 

 a separation in the case of the aerobes. We have therefore investigated 

 other inhibitors to see if we could find some substance which would 



