15. D. Davis: Studi 



Nutritionally Deficient Bacterial Mutants Isolated by Means of Penicillin 



known in microbiology as the satellite phenomenon; 

 systematically employed, it has been our vade-mecum. 



Mutants obtained 



Mutants of E. colt ("Waksman" strain 1 , Amer. Type 

 Culture Collection 9637) have been obtained with re- 

 quirements for all the naturally occurring amino acids 

 except alanine, aspartic acid, and hydroxyproline. A 

 number of mutants respond to either serine or glycine; 

 thus far, none of our strains has been specific for either 

 of these interconvertible amino acids. The sulfur- 

 deficient mutants, which are a very common class, 

 respond to cystine or less rapidly to methionine, and 

 are blocked in the reduction of sulfate to sulfite or of 

 sulfite to thiosulfate or sulfide. In addition, there are 

 mutants, unresponsive to thiosulfate or sulfide, with 

 specific requirements for cystine and others for 

 methionine. Besides specific proline-requiring mutants, 

 there are others which respond to either proline or 

 glutamic acid or a-ketoglutaric acid (but not ornithine) . 



Many of these types of mutants have already been 

 isolated from E. coli by earlier techniques 2 . In addition, 

 we have isolated strains with more complex require- 

 ments which should throw light on certain metabolic 

 relationships. Alternative requirements exist for lysine 

 or threonine, and, in another mutant, for a-amino 

 butyric acid or isoleucine (or, curiously, D-threonine 

 but not L-threonine). One peculiar mutant responds 

 either to methionine or to thiamine: to methionine in 

 the concentrations of several micrograms per ml usual 

 for amino acid mutants; to thiamine or its pyrimidine 

 in the concentrations, one thousandth as great, re- 

 quired by other thiamine mutants. Another mutant 

 similarly requires, under special conditions, either 

 methionine or vitamin B-12. Finally, there are several 

 mutants with a multiple requirement apparently due 

 to a single genetic block: isoleucine plus valine; 

 phenylalanine plus tyrosine; phenylalanine plus 

 tyrosine plus tryptophan; and these three aromatic 

 amino acids plus />-amino benzoic acid. No peptide- 

 requiring mutants have been obtained; although much 

 of our isolation work has been done with tryptic casein 

 hydrolysate, which contains many peptides, all the 

 mutants isolated from this enrichment have grown on 

 a mixture of known amino acids. One serine or glycine 

 mutant and one methionine mutant, like several re- 

 ported Neurospora mutants, are temperature sensitive, 

 with an absolute requirement at 37°C, and none at 

 25°C. 



With yeast extract or hydrolysed yeast nucleic 

 acid, mutants have been obtained with requirements 



1 The initiation of this work with the "Waksman" strain has 

 In en accidental. For any new program, it would undoubtedly be 

 preferable to use the K-12 strain of E, coli, with which genetic 

 rei ombination can be studied 3 . 



- E.L. Tatum, Cold Spring Harbor Symp. Quant. Biol. n, 278 

 (1946). - B. I). Davis, Arch, liiochem. 20, 166 (19491. 



:l ]■;. I.. Tatum, and .]. Li: of rrf.ro, J. Bact. S3, 673 (1947). 



for purines or pyrimidines. The purine mutants, 

 however, have all responded to adenine or guanine or 

 hypoxanthine or their ribosides or nucleotides, while 

 several have also responded to xanthine ; the pyrimidine 

 mutant has responded to cytosine or thymine or uracil 

 or their ribosides or nucleotides. Because of this non- 

 specificity, this group has not been further studied. 



Among the vitamins, mutants have been obtained 

 with individual requirements for thiamine, nicotin- 

 amide or nicotinic acid, pyridoxin or its amine or 

 aldehyde, />-amino benzoic acid (PABA), pantothenic 

 acid, and biotin. In spite of a number of attempts, none 

 have been obtained with requirements for riboflavin, 

 inositol, choline, or hemin. The reason for these 

 failures is not apparent. One mutant requiring an 

 unknown factor in yeast extract has been obtained 

 twice. 



Microbiological assay 



We have not engaged in extensive studies on the use 

 of these mutants for microbiological assay. Since the 

 medium employed is so simple, mutants might be 

 expected to have some advantage over the wild-type 

 species with complex requirements which are generally 

 used. In addition, turbidimetric assay of bacterial 

 growth is more convenient than the measurement of 

 dry weight of pellicle which is required with Neuro- 

 spora mutants 1 . The advantage of the mutants would 

 be largely lost, however, if a heavily enriched medium 

 were required in order to prevent other substances 

 present in the material under assay from altering the 

 quantitative response to the required nutrilite. Since 

 secondary effects of other substances have been 

 observed in many instances with wild-type organisms, 

 it would be necessary with each mutant to determine 

 the conditions for assay. Furthermore, the instability 

 of most of the mutants requires caution. With several 

 mutants of ordinary stability (phenylalanine, tyrosine) 

 we have been able to obtain satisfactory growth curves, 

 without significant appearance of reversions, at 24 

 hours, but by 48 hours irregular increases in turbidity 

 due to reversions had appeared. 



Syntrophism 



The syntrophic technique is illustrated with three 

 arginine-requiring mutants which are shown in Fig. 1 

 to be blocked at different stages in the well-known 

 Krebs-Henseleit scheme: ornithine-x:itrulline->-argi- 

 nine. Sets of mutants of both Neurospora and E. coli, 

 blocked at these stages, had previously been reported 2 , 

 having been recognized by their response to these 

 precursors of arginine, In the case of growth factors 

 whose precursors are unknown, however, and which 



1 F. J. Ryan, Feder. Proc. S, 366 (1916). 



8 A. M. Srb, and N. H. Horowitz, J. Biol. Chem. 154, 129 (1944). 

 R. R. Roepke, quoted by E. L. Tatum, Cold Spring Harbor Symp. 

 Quant. Biol. 11, 278 (1946). 



9 6 



