44 



B. D. Davis: Studies on Nutritionally Deficient Bacterial Mutants Isolated by Means of Penicillin [Experientia Vol. VI/2] 



are therefore the most interesting, this technique of 

 substituting precursors cannot be used, except by 

 guesswork, to recognize differences in the site of 

 genetic blocks. For this reason, the alternative tech- 

 nique of syntrophism is particularly useful. This 



Fig. 1.- Growth of mutants and wild-type (S stock) on minimal 

 medium enriched with ornithine, citrulline, or arginine. It is seen 

 that one mutant responds to any of the three related compounds, 

 another to either citrulline or arginine, the third to arginine only. 



technique is shown in Fig. 2, in which the mutant 

 specifically requiring arginine is seen to excrete a 

 factor, presumably citrulline, which stimulates the 

 growth of the two mutants blocked earlier ; while the 

 citrulline mutant in turn feeds the ornithine-requiring 

 mutant. The gradient of observed growth reflects the 

 gradient of diffusion through the agar. 



Similar relationships, involving unknown precursors, 

 have been observed among proline and among histidine- 

 requiring mutants and among certain mutants requir- 

 ing aromatic amino acids. Syntrophic accumulation of 

 precursors appears to be quite widespread with E. coli 

 and has been observed among sets of mutants (e. g. 

 arginine) which fail to show accumulation in Neuro- 

 spora. Its absence between two mutants with a com- 

 mon requirement, however, does not prove that they 

 are blocked at the same enzymic site, since precursors 

 may fail to accumulate because of instability, diversion 

 along an alternative path, or the inability of the cell 

 to build up a concentration adequate for excretion. 



Causes of syntrophism 



Since syntrophism leads to recognition of the ac- 

 cumulation of a precursor whose subsequent isolation 

 and identification will contribute to the analysis of a 

 biosynthetic chain, it becomes important to be aware 

 of other possible sources of this phenomenon. Several 

 have so far been observed. 



(1) Accumulation of the precursor of a genetically 

 blocked reaction. This mechanism is the one discussed 

 above. 



(2) Excretion of metabolites by wild-type E. coli. It is 

 known that bacteria not only remove nutrilites from 

 the medium, but contribute metabolic products to it. 

 In order to find whether these include growth factors 

 for any available mutants, the wild-type strain was 

 streaked on minimal medium adjacent to mutants with 

 single requirements for each of the factors listed above. 

 After 48 hours, three of the mutants were so heavily 

 fed as to produce maximal growth. These were the 

 strains requiring biotin, PABA, and pantothenic acid. 

 In addition, the nicotinamide-less mutant was moder- 

 ately fed. The other available vitamin-requiring 

 mutants (thiamine and pyridoxin), as well as a purine, 

 a pyrimidine, and all the amino acid-requiring mutants, 

 showed that the amounts of their required factors 

 excreted by wild-type were negligible, supporting at 

 most only microscopic growth after 2 to 5 days. It is 

 concluded that this strain of E. coli is economical in its 

 synthesis of all the growth factors that could be tested 

 except four vitamins. It is of interest to note, however, 

 that the amounts of the other factors excreted, at 



Fig.. 2. - Syntrophism among arginine-requiring mutants of Fig. 1. 

 Mutant responds to ornithine or citrulline or arginine; C responds 

 to citrulline or arginine; A responds to arginine onlv. 48 hours of 

 growth (37° C) on medium enriched with very small amount of casein 

 hydrolysate ("NZ Case"). 



97 



