pathway; in the absence of bicarbonate, they 

 developed along the OC path. Thus, when this 

 work began, we set out to learn by what means 

 this bicarbonate trigger mechanism brought 

 about the de novo synthesis of melanin and 

 carotene, the increased rate of synthesis of 

 chitin, and the numerous other characteristics 

 associated with the structure and function of the 

 resistant sporangial cell. 



In the late 1950's, mainly through the skill- 

 ful and conscientious efforts of my former 

 students - Drs. James Lovett, HowardMcCurdy, 

 Evelyn Horenstein and others - methods were 

 envolved for growing submerged, uniformly 

 distributed cells of the fungus along one path- 

 way or the other in massive, synchronized, 

 single-generation cultures. By starting with 

 several hundred million or a billion spores, all 

 of them of exactly known age, such spores were 

 made to go through their acts in nearly perfect 

 synchrony along either path. Thus, it becomes 

 immediately evident that, by this means, one 

 can harvest almost any number of OC cells all 

 of which have arrived at, let us say, 30% of 

 their exponential period of growth; or OC cells 

 all of which are tooling up to manufacture a 

 second generation of spores; or a billion RS 

 cells all of which are in the process of laying 

 down the cross wall which will delimit the 

 resistant sporangium from the rest of the thallus, 

 and so on. Here, then, was an elegant system for 

 studying the relations between biochemical and 

 morphological differentiation, and we began tc 

 exploit it. 



TILL: Have you got the time scale on this? 



CANTINO: The time scale for the OC path- 

 way depends upon the nature of the medium and 

 other factors. Under the conditions we have used, 

 it ranges from 12 to 17 hours, depending on popu- 

 lation density. For the RS pathway, our standard 

 procedure yields a generation time of 84 hours. 



TILL: The other point I'd like to ask about 

 is, are those spores the same? 



CANTINO: Morphologically, they appear to 

 be identical. (Note added in proofs: It should be 

 pointed out that all of our work with synchro- 

 nized liquid cultures is done with spores derived 

 from OC cells. The reason for this is that it is 

 a simple matter to prepare massive suspension 

 of such spores, but a much more difficult one 

 to prepare them from RS cells. There is some 

 evidence, however, that the developmental po- 

 tentials of - i.e., the kinds of progeny produced 

 by - RS-spores and OC-spores are not identi- 

 cal (D). 



Now, let us come to grips with the nature 



of this bicarbonate trigger mechanism. This 

 has been under investigation in my laboratory 

 for a long time. Therefore, let me simply pre- 

 sent a condensed recapitulation of the essential 

 biochemical event which we think is operative; 

 then, we can use this as a convenient point of 

 departure. At the metabolic level, the focal point 

 appears to be as follows. In homogenates of OC 

 cells of various ages, the enzymatic activities 

 of the tricarboxylic acid cycle are detectable; 

 thus, the cycle is at least potentially functional. 

 In spores and germlings of such OC cells, the 

 in vivo evidence suggests that the Krebs cycle 

 is operating. In Fig. 3, on the left, only one of 

 the steps in the cycle is shown - the step medi- 

 ated by a TPN-specific isocitric dehydrogenase. 

 But, when bicarbonate is added to a spore or 

 developing germling, it quickly induces a mul- 

 tiple set of enzymatic lesions in the tricarboxylic 

 acid cycle. However, the isocitric dehydro- 

 genase remains functional, and it now begins to 

 operate in reverse, mediating reductive car- 

 boxylation of ketoglutarate back to isocitrate 

 (Fig. 3, right). At the same time, bicarbonate 

 also induces the formationofisocitratase, which 

 cleaves the isocitrate to succinate and glyoxy- 

 late and thus prevents its accumulation. Finally, 

 a constitutive glycine-alanine transaminase in 

 the organism helps to keep the chain of reac- 

 tions on the move by amination of glyoxylate to 

 glycine at the expense of alanine. 



Let me illustrate, by way of a few examples, 

 some of the kinds of information obtainable 



«n 



f 



OC Plortt 



PYH. 



iLYCI 



(t 



■'ALANINE 



GLYOX. 



+ 



sue. 



V 



/, 



GLYOX 



sue 



'> 



■•■^ 



. ISOCITR. 



y 



TPNH 



Fig. 3. 



The bicarbonate trigger mechanism in Blastocladiella 

 emersonii. (Fig. 6, Cantlno, In "11th Symp. of the Soc. 

 for Gen. Microbiol.", 1961; reproduced with permission 

 of the Society for General Microbiology.) 



151 



