Studies on Nutritionally Deficient Bacterial Mutants 

 Isolated by Means of Penicillin 1 



By Bernard D. Davis 2 , New York, N.Y. 



Studies on the mode of action of chemotherapeutic 

 agents have not shown any direct effect on the known 

 catabolic processes of bacteria. There is good reason, 

 by exclusion, to suppose that they act by means of 

 interfering with anabolic reactions, about which much 

 less is known. In the last decade, Beadle and his 

 school have made striking contributions to this area in 

 biochemistry, as well as to genetics, by the use of 

 nutritionally deficient ("biochemical") mutants of the 

 mold, Neurospora -mutants which have lost one or 

 another specific enzymic activity 3 . More recently, 

 bacteria have been found to yield mutants of the same 

 sort 4 . These microbial mutants offer a promising 

 approach to the major theoretical problems of chemo- 

 therapy: the mechanism of chemotherapeutic action 

 and the biochemical alterations responsible for drug- 

 resistance in microorganisms. Furthermore, the pheno- 

 menon of drug-resistance presents a direct problem in 

 microbial genetics. Finally, just as the nutritional 

 requirements of various wild-type microorganisms have 

 been of tremendous value in isolating the known 

 vitamins, so the extension of this approach to the 

 induction of artificial nutritional requirements offers 

 promise as a method for detecting metabolites peculiar 

 to microorganisms -metabolites of special promise as 

 models for the construction of inhibitory analogues. 

 For these several reasons this laboratory, interested in 

 chemotherapy, has felt warranted in undertaking an 

 excursion into microbial genetics. 



It is a simple matter to isolate, from huge microbial 

 populations, rare mutants that can survive or prolifer- 

 ate in a medium which suppresses the parent strain. 

 Examples include mutations to drug-resistance, to 

 bacteriophage-resistance, or to decreased nutritional 

 requirements. Comparable techniques have not been 

 available for the nutritionally deficient mutants, 

 though these are in some respects more interesting. In 



1 Based on material delivered in a symposium on The Relation 

 of Genetics to Biochemistry at the annual meeting of the American 

 Chemical Society in San Francisco, Cal., March 30, 1949. 



2 U. S. Public Health Service, Tuberculosis Research Laboratory, 

 ( ornell I iniversity Medical College, New York -21, X. Y. - This paper 

 is in the hands of the editor since [une 16th, 1949, 



3 G. W. Beadle, Physiol. Rev 2S, 643 (1945); Chem. Rev. 

 :i7, 15 (1945). 



4 E.L.TATUM,ColdSpringHarborSymp.Quant.Biol.lJ,278(1946). 



the early investigations, deficient mutants were isol- 

 ated by testing of spores or colonies selected at random-- 

 a tedious process, since the total frequencv of recogniz- 

 able mutants, even after optimal irradiation, rarely 

 exceeds 1 or 2 per cent. Improved techniques of isola- 

 tion have recently been introduced for Neurospora 1 

 and other molds 2 , and for bacteria 3 . In the case of the 

 bacteria, the techniques depended upon delayed or 

 limited enrichment of the medium, with consequent 

 production of small colonies of mutants which could be 

 distinguished from the larger colonies of the parent 

 strain. Since the colonies must not be too crowded, 

 these techniques were limited to a few hundred colonies 

 per Petri dish. 



The penicillin method 



For the isolation of rare mutants, a method permit- 

 ting selection from much larger populations would be 

 desirable. The possibility of such a method suggested 

 itself on the basis of the report that penicillin has the 

 remarkable property of sterilizing ("killing") bacteria 

 only under conditions which permit growth 4 . We have 

 confirmed and extended this conclusion. In a minimal 

 medium containing glucose, lactate, ammonia, and 

 sulfate as sole sources of carbon, nitrogen, and sulfur, 

 various amino acid-requiring mutants of E. coli are 

 completely resistant to the bactericidal action of 

 penicillin. The addition of the required amino acid, 

 however, renders the mutant as sensitive to penicillin 

 as its parent wild-type strain. In applying this method 

 to the isolation of new mutants, a suspension of E. coli 

 was irradiated with ultraviolet light, further cultivated 

 in an enriched medium, washed, and then incubated, 

 in inocula of suitable size, with penicillin in. minimal 

 medium for 24 hours. Up to 100 p. c. of the large num- 

 ber of survivors, recovered by plating in agar media 

 supplemented with casein hydrolysate or yeast extract, 

 were found to be nutritionally deficient mutants. The 

 penicillin method has been independently developed in 



1 J. Lein, H. K. Mitchell, and M. B. Houlahan, Proc. Nat. 

 Acad. Sci. 34, 435 (1948). 



2 N. Fries, Nature 159, 199 (1917). 



3 J. Lederherg, and E. L. Tatim, J. Hid. Chem. 165, 3S1 

 (1946). - B. I). Davis, Arch. Biochem. 20, 166 (1949). 



4 ('.. I.. Hobby, K. Meyer, and E. Chaffee, Proc. Soc. Exp. 

 Biol. Med. 50, 281 (1942). 



[Reprinted by permission of Vcrlag Birkhauser, Basel, from Expi rii ntia 6 : (2) 41-50, 1950] 



94 



