Dec., 1948
COMMUNICATIONS TO THE EDITOR
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factor which limits the survival of mutants is the syntrophic effect of metabolites secreted by the non-mutated cells growing in minimal medium. ALFREY, JR. INSTITUTE OF POLYMER RESEARCX TURNER The density of the population exposed to penicillin POLYTECHNIC INSTITUTE OF BROOJCLYN BROOKLYN, NEWYORK HARRY WECHSLERis therefore best limited to lo6cells/ml. JUNE 10, 1948 RECEIVED By this technique mutants of E. coli (“Waksman” strain, ATCC 9637) have been obtained with individual or alternative requirements for all ISOLATION OF BIOCHEMICALLY DEFICIENT the naturally occurring amino acids except alaMUTANTS OF BACTERIA BY PENICILLIN nine and hydroxyproline; for several multiple sets Sirs: of amino acids; for purines or pyrimidines and It is possible to isolate bacterial mutants with their derivatives; for most vitamins; and for unease when the mutants can proliferate or survive known factors in yeast extract. in an environment in which these activities are This procedure should make i t possible for bionot possible for the parent strain. There is chemists to isolate desired types of mutants a t will. therefore no difficulty in obtaining mutants, even These mutants, which have some advantages over of low frequency, which differ from the parent Neurospora, can be used for not only quantitative strain by resisting bacteriophage or antibacterial but also very simple qualitative microbiologic aschemicals, or by having decreased nutritional re- say, as well as for discovery of new metabolites, quirements. Mutants with increased nutritional and production of rare chemicals by mutants requirements, however, though a class of espe- which accumulate the substrate of the blocked encially great biochemical interest, have been much zymic reaction. A more detailed account is being less convenient to isolate. Recently developed published.& permit a considerable improvement STATES PUBLICHEALTH SERVICE over the earlier practice of random selection, but UNITED TUBERCULOSIS RESEARCB LABORATORY still permit selection from only a few hundred CORNELL UNIVERSITY MEDICAL COLLEGE colonies per agar plate. NEWYORK 21, N. Y. BERNARD D. DAVIS The possibility of isolating these biochemically RECEIVED DECEMBER 1, 1948 deficient mutants from much larger populations suggested itself on the basis of the reports3e4that CONCENTRATION OF BIOCHEMICAL MUTANTS OF penicillin sterilizes only growing bacteria. We BACTERIA WITH PENICILLIN‘ confirmed this conclusion, and found that a tryp- sir: tophan-less mutant of E . coli was completely reExisting methods for isolating biochemical musistant to the bactericidal action of penicillin in tants are still tedious, although mitigatory prominimal medium unless tryptophan was added. cedures have been described.2 We have found The technique was successfully applied to the that penicillin can be used to augment the proporisolation of new mutants. Ultraviolet irradiated tion of mutants in a culture, greatly facilitating bacteria were cultivated overnight in medium en- their isolation. riched with casein hydrolysate, washed, and exThe method depends on the finding that peniposed to penicillin (300 O.U./ml.) in minimal cillin lyses only growing cells with little permanent medium6 for 24 hours. Large numbers of colonies effect on resting suspensions.a This suggested (ca. 100, from an inoculum of los bacteria exposed that, if allowed to act on a mixture of mutant and to penicillin) were isolated on enriched agar; over non-mutant cells in a synthetic medium, peniS070 were mutants. These include replicates aris- cillin might concentrate the mutants which are ing from each original mutant during intermediate unable to grow in this medium. cultivation; a variety of types, however, can be These expectations were first tested in reconrecovered on a single plate. struction experiments. Y-53 is a mutant of I n earlier experiments bacteria had been exposed Escherichia coli requiring threonine, leucine and to penicillin following irradiation, without inter- thiamin, and is lactose-negative; K-12 is its lacmediate cultivation; no mutants were obtained. tose positive wild type ancestor. Suspensions This failure depends on a lag in the adjustment of were assayed for mutants by planting on EMBthe enzymic composition of the cell to the new ge- lactose agar4 and counting the dark and light nic composition. Until the cell has gone through colonies as K-12 and Y-53, respectively. After enough generations to dilute out the enzyme preliminary study of various conditions, the folmolecules which were formed by the gene prior to lowing were adopted : Washed suspensions of its mutation, the cell does not lose its capacity to young cells harvested from a complete medium form a given metabolite, and hence is not resist(1) Supported by p n t s from the Diviaion of Research Grants and ant to penicillin in minimal medium. Another Fellowships of the Nation81 Institute of Health, U. S. Public Health
attributed to the electron-withcl-zwing character of the -COOR group.
(1) J. Lederberg and E. L. Tatum, J . Bioi. Chum., 16S, 381 (1946). (2) B. D. Davis, Arch. Biochcm., in p r e s .
( 3 ) G. L. Hobby, K. Meyer and E. Chaffee, Proc. B i d . Med., 60, 281 (1942). (4) E. Chain and E. S. Duthie, Lancd. 1, 662 (1945). c.5) B. D. Davis, Proc. N o f . Acad. Sci.. in press.
oc. Ergl.
%vice, and the Rockefeller Foundation and The Wisconsin Alumni Research Foundation. Paper 984, Department of Genetics, College of Agriculture, University of Wisconsin. (2) J. Lederberg and E. L. Tatum, J . Bioi. Chcm., 165, 381 (1946). (3) G. L. Hobby, Ptoc. Soc. Ezptl. Biol. Med.. 66, 181 (1944). (4) J. Lederberg, Genetics, SI, 505 (1947).
