ketoglutaric dehydrogenase increase only 6t)0 

 timesi Thus, the 90% decrease in oxygen con- 

 sumption goes hand in hand with the 90% de- 

 crease in the intracellular accumulation of 

 ketoglutaric dehydrogenase relative to the iso- 

 citric dehydrogenase which immediately pre- 

 cedes it in the Krebs cycle. It appears, there- 

 fore, as if bicarbonate causes the ketoglutaric 

 dehydrogenase system to become a bottleneck 

 in the cycle, that it begins to do so early in 

 ontogeny, and that this soon brings the activity 

 of the tricarboxylic acid cycle to a halt. 



What about the other critical enzyme in the 

 scheme - the isocitratase? Some years ago. Dr. 

 McCurdy purified it and established its proper- 

 ties. Assays with synchronized cultures show 

 how it, too, is involved. Figure 6 reveals what 

 happens to the total units of isocitratase per 

 cell during development in the presence and 

 absence of bicarbonate. The intracellular quan- 

 tity of this enzyme in the spore is shown on the 

 vertical axis, i.e., at zero time. As the spore 

 gives rise to a germling, and it in turn develops 

 exponentially into a young OC plant in the ab- 

 sence of bicarbonate (bottom curve), there is 

 no net synthesis of this enzyme. It seems as if 

 the original amount of isocitratase in the spore 

 is simply diluted out as the growing cell in- 

 creases in size. Only when the OC cell has 

 reached about half of its generation time does 

 synthesis of isocitratase begin. However, when 

 spores are germinated in bicarbonate media, 

 exponential synthesis of isocitratase apparently 

 begins immediately (upper curve). In summary, 

 while bicarbonate brings about a lesion in the 

 tricarboxylic acid cycle by creating a bottleneck 

 at the locus of ketoglutaric dehydrogenase, it 

 provides relief for the damage done by inducing, 

 simultaneously, synthesis of isocitratase. Thus, 

 in the bicarbonate-induced RS cell, isocitrate 

 leads to succinate and glyoxylate (and thence to 

 glycine), whereas in the bicarbonate -independent 

 OC cell, it leads to ketoglutarate (and thence 

 to succinate) and CO2 . 



Finally, let me present one last set of data 

 which bear upon this mechanism. If the pro- 

 posed scheme is correct, in vivo uptake of 

 CO 2 and/or bicarbonate by a developing RS 

 cell should reach its peak at that point in 

 ontogeny where the cell's complement of iso- 

 citric dehydrogenase is maximum relative to 

 its complement of the bottleneck enzyme, keto- 

 glutaric dehydrogenase (this point in ontogeny, 

 as will be seen in a subsequent figure, occurs 

 at about 36 hours, i.e., 43% of the RS cell's 

 generation time). To test this notion, RS cells 



were grown in synchronized culture and then, 

 at 30 hours, provided with a dose of H^^COs 

 and allowed to continue growing for 6 hours. 

 During this latter period, cells were sampled 

 and assayed for total ^''C fixed, and the medium 

 assayed for total i^C which had disappeared. 

 A similar experiment was done in which 38 hour 

 cells were fed the H^'^COs. The results were 

 combined to yield one graphs as shown in Fig. 7. 

 You will note that uptake of H^^COg per cell 

 increases as the cell passes through 36 hours 

 of age, and that uptake decreases again after 

 39-40 hours. Since the ratio of total units of 

 ioscitric dehydrogenase to units of ketoglutaric 

 dehydrogenase is maximum at 36 hours, and then 

 decreases once again beyond this point, these 

 data provide further evidence that the bicarbon- 

 ate trigger mechanism operates as proposed 

 above. 



Let us move on, now, to consider the transi- 

 tion period between exponential growth of the 

 RS cell and its subsequent differentiation; the 

 data I wish to present in this connection also 

 have a direct bearing upon the biochemical 

 mechanism we have been discussing. The photo- 

 graphs which are shown in Fig, 8 were taken 

 by Dr. Lovett when he was working in my lab, 



V, Gen. Time 



Fig. 6. 



Synthesis of isocitratase during growth of OC and RS 

 cells. (Fig. 5, Cantino, In "11th Symp. of theSoc.for Gen, 

 Microbiol,," 1961; reproduced with permission of the 

 Society for General Microbiology.) 



153 



