54 Perspecfives in Microbiology 



by using enzymatic methods alone, or even in combination 

 with isotopic methods. Let us consider the tricarboxylic 

 acid cycle, whose energy-yielding, degradative aspects have 

 been emphasized in the past, though its importance in the 

 biosynthesis of glutamic acid and aspartic acid and their 

 derivatives has only recently received more attention. De- 

 spite the fact that all the enzymes of this cycle have been 

 demonstrated in E. coli and in various other micro- 

 organisms, there is no general agreement as to whether this 

 cycle plays an essential or even an important role in the 

 oxidation of acetate by these organisms. 



To study this question, Gilvarg and I have used the 

 methods described for biosynthetic reactions, employing a 

 glutamic acid auxotroph of E. coli that turned out, on 

 enzymatic analysis, to lack the condensing enzyme for form- 

 ing citrate from acetyl-coenzyme A and oxaloacetate (14). 

 It was found that this mutant not only required a-ketoglu- 

 tarate or glutamate as a growth factor (when growing on 

 glucose as a carbon source), but also had completely lost the 

 ability to use acetate as a carbon source or to form CO2 from 

 acetate. Yet when E. coli derives its energy from acetate, 

 the formation of CO2 is obligatory.^ Hence, in terms of the 

 criteria proposed, we have shown that absence of the citrate- 

 condensing enzyme is necessarily associated with inability 

 to form from acetate two obligatory metabolic products 

 (a-ketoglutarate and CO2) which the wild type can form. 

 We can therefore conclude that the tricarboxylic acid cycle 

 is essential in E. coli not only for biosynthetic purposes, but 

 also for the complete oxidation of acetate. I believe this is 

 the first conclusive demonstration of the latter point; it 

 provides an excellent example of the sharpness of the tools 

 furnished by microbial mutants. 



5 The possibility of succinate formation by back-to-back condensation 

 of acetate was eliminated by the fact that this mutant, though unable 

 to oxidize acetate, could still oxidize succinate. 



