Microbial Mutants 41 



in turn, were based only on indirect, nutritional evidence. 

 I should therefore like to describe the results of enzymatic 

 studies that my colleagues and I have undertaken on ex- 

 tracts of a variety of mutants. Furthermore, I should like 

 to consider in some detail the biochemical significance of 

 these findings, for they permit us to judge, much more 

 rigorously than before, whether compounds revealed by 

 nutritional and accumulation studies are true, normal 

 metabolic intermediates.^ Finally, I shall point out how 



1 1 should like to propose the following definitions: 



An obligatory intermediate is a member of a path that is the only one 

 by which an organism can synthesize a given product at an adequate rate 

 from given source materials. Most of the known biosynthetic paths be- 

 long to this category, since a block in almost any one of them gives rise 

 to a growth requirement. 



It should be emphasized that obligatory intermediates must be defined 

 with respect to the beginning as well as the end of the metabolic path 

 under consideration. Thus, in various organisms growing on glucose and 

 ammonia alone, it is well known that cystathionine and homocysteine are 

 obligatory precursors of methionine. Replacement of the glucose by other 

 general carbon sources, such as other carbohydrates, acetate, or glycerol, 

 would not be expected to change this path. Addition of the a-keto acid 

 corresponding to methionine, however, could produce such a change; 

 for though this compound is not on the path between carbohydrates and 

 methionine, it can be nonspecifically transaminated by some organisms 

 to form methionine. In consequence, cystathionine and homocysteine 

 would no longer be obligatory intermediates when an organism that 

 could transaminate this keto acid at a sufficient rate was grown in a 

 medium containing an excess of the compound. In some analogous cases, 

 it has been demonstrated by the method of isotopic competition that the 

 presence of certain precursors of amino acids can completely eliminate 

 the formation of those amino acids from general carbon sources and 

 hence completely eliminate the participation of the earlier intermedi- 

 ates (1). 



We shall consider an essential intermediate to be one that is obligatory 

 on a minimal medium (that is, a medium containing only general carbon 

 sources, any growth factors required by the wild type, and inorganic 

 salts). 



If the term normal intermediate is to have any useful meaning, it seems 

 necessary to restrict its application to the use of source materials that 

 are defined as normal; otherwise all metabolites produced by an organism 

 would be normal, and all those that could be incorporated into some 

 metabolic product would be normal intermediates. I would suggest that 

 a normal medium be considered synonymous with the minimal medium. 

 Then on a biosynthetic path for which there is no alternative, a normal 

 intermediate would be synonymous with an essential intermediate. Where 

 there are minor alternative paths (as in the formation of valine by 



