48 Perspectives in Microbiology 



Furthermore, it is not a sufficient condition. For example, 

 para-nitrobenzoic acid can effectively replace para-amino- 

 benzoic acid as a growth factor for mutants of Neurospora 

 (29) or E. coli (10), but only because the cells can non- 

 specifically reduce nitro groups to amino groups. In such 

 a case, even if one used isotopes to show that growth factor 

 A was incorporated into cell constituent B, one would have 

 demonstrated only that A can serve as a precursor of B, 

 but not that A is an essential precursor of B, that is, an 

 obligatory intermediate in the synthesis of B by the wild- 

 type organism growing on a minimal medium. 



Accumulation of a compound by an auxotrophic mutant 

 has seemed to offer more significant evidence that the com- 

 pound is a true intermediate. But this property, too, is 

 clearly not a necessary condition.^ Furthermore, it is not 

 a sufficient one. Some accumulated compounds are unstable 

 and give rise, for example, to colored products that are 

 very unlikely to be participants in biosynthesis (3, 21); and 

 some accumulated intermediates appear to become con- 

 jugated, in a process akin to what in mammalian biochem- 

 istry is called detoxification (33). 



We come, then, to enzymatic criteria. If a compound has 

 nutritional activity, it must either be, or become spontane- 

 ously converted to, the substrate of some enzyme in the 

 cell. Similarly, if a compound is accumulated, it must 

 either be the product of some enzyme or be spontaneously 

 formed from such a product. In consequence, the demon- 

 stration of such an enzyme does not alone, any more than 



2 For example, when an intermediate is common to several paths (as 

 with glutamic acid), a block in its entrance to one of those paths would 

 not be expected to cause its accumulation. Even with compounds re- 

 stricted to a single path, the level of accumulation reached varies widely 

 from one compound to another and also depends strikingly on the con- 

 ditions of growth; it is sometimes not detectable. In general, accumulation 

 of intermediates by mutants has been used by students of intermediary 

 metabolism simply as a generous gift of the gods. The mechanisms 

 responsible for such accumulation and, even more important, for its 

 absence in the wild type constitute a major problem for future investiga- 

 tion. 



