THE UNIVERSITY OF TEXAS AUSTIN 12 DEPARTMENT OF BOTANY AND BACTERIOLOGY June 2, 1948 Dr, Joshua Lederberg The University of Wisconsin College of Agriculture Department of Genetics Madison 6, Wisconsin Dear Dr, Lederberg: I have instituted a search for the copy of the M.A. thesis material in your letter of May 20 and have not as yet been able to find my copy. I suspect someone has bor- rowed it but it should show up. I find that the most in- teresting experiments were done after it was written and here is the essence of the thing. Micrococcus aureus strains resistant to penicillin were grown together with similar cultures resistant to streptomytin. The cultures were also grown separately. After 5 or 6 hours incubation the cultures were plated to determine total numbers and also the proportion of each mutant present and the number that were resistant to both antibiotics. A typical result was: No, of organisms per cc from plate counts. Penicillin Streptomycin Mixed mut ant mutant culture Nutrient agar 60, 000,000 80, 000, 000 80, 000, 000 Nutrient agar + penicillin 48,000, 000 28 22,000, 000 Nutrient agar + streptomycin 210 60, 000, 000 30,000, 000 Nutrient agar + both’ 100 12 4, 000 The irradiation experiments involving the E. coli recom- binations will appear in the next issue of the Proc, Nat. Acad, Sci, I believe that we discussed the details, I am afraid my lack of a good genetic background prevents me from seeing how a recombination of lethal factors could be involved in the haploid except by some very tricky mechanism for which there is as yet, no evidence, I enclose a rough draft of a talk I gave about a year ago on E, coli transmutation, The abstract of this is pub- lished in J, Bact. We wasted some time on patent possibili- ties of the practical aspects of this work and the status of the problem is about where it was a year ago, The new stu- dents working on the problem have made little progress and are now attempting to isolate new strains for use in this work, Kindest personal regards Ql Moe OW/pam Enel, Many antibacterial agents are unsuitable inhibitors because of the resise tant individuals which cccur as spontaneous mutations in the microbial pepula- tion, These individuals grow in the presence of the inhibiter and establish a resistant culture against which the inhibiter is ineffectual, We have devoted considerable study to the occurrence of such individuals, The recent work en die rected icutations suggested thet it may be possible to modify the reskstance of a bacterial pepulation by use of such techniques, The work at the kockefeller In« stitute on the trensformatien of the pneuncceccus and the studies ef Bewin and collaborators (ce pte, rend. sec, biel, ad2, 1047, 1945) have shown that certain charact+ristics of a bacterial pepulation can be modified. by the use of a thyme nucleic acid extract ef ancther Strain, If this methed of inducing mutations is a general one it might be put tc practical use in the field :f bacterial resis tance to inhibitory agents, These studies were made with eur laboratory strain of Ee GQli (designated as the sensitive strain) end a sulfonamide resistant strain develeped frem it, A quantity of celis of beth strains was harvested and extracts frem each wore prepared, The finel precedure adepted is reported in table 4, but the early studies were made with the amide extracts and with the nucleepretein rather than with the purifiea nucleic acids, These neterials were sterilized by per mitting them to stand under 70% ethanel evernight, They were then removed from the alcohel, dissolved in sterile buffer, and added te a series of test tubes which contained a synthetic J. S014 medium and several concentrations ef sulfa- nilamide, The experiment was set up in triplicate with one series centaining ne nucleepretein extract, a second containing extract frex the resistent strain and a third with extract from the sensitive Strain, Sterility centrels for the ex- tract were included and al. the ether tubes were ineculated with a srall inecu- lum frem a yeung culture ef the sulfenamide resistant strain, The results in table 1 shew thet the resistant c lture grew slightly in the = 2 =/ Cohtinued presence of 30 mg % sulfanilamide and that the addition to the medium of an ex~ tract of the resistant culture had no effect on this resistance, The addition of an extract from a sensitive culture, however, prevented the growth by 30 mg% and even by 20 mgé of sulfanilamide, The experiment reported in table 2 was set up in an identical manner but was inoculated with the sensitive strain of E. coli. It is observed that this or ganiam is completely inhibited by 5 mg% of sulfanilamide both in the control series and where an extract of the sensitive strain was added to the medium, The addition of extract from the resistant strain permitted slight growth even in 20 mgs of sulfanilamide, A study of the quantitative changes in the popuiation is reported in table 3, Here the organisms were grown in the presence or absence of the bacterial extracts as indicated but without any sulfanilamide added, After 24 hours the mature cultures were plated in agar containing varying amounts of sulfanilemide to determine the distribution of resist:nee in the resulting population, as can be seen from the plate counts the presence of an extract from a resistant culture increases the average resistance of a sensitive popuiation, And more surprising the presence of extract from sensitive cells decreased the average resistance in the resistant population, An experiment utilizing the purified nucieic acid extracts prepared as ine dicated in table 4 showed that these contained the "transforming principle", The most obvious explanation of this conception assumes that mutations re~ sult from inexact replications of the genetic mechanism of the microbe and that these “errors” are found in the nucleic acid, Under some conditions or- ganisms may assimilate these preformed fragments of their genetic control mechanism if they are added to the medium, If the fragments are slightly dif- ferent from those ordinarily tmilt by the organisms the result is a mutation, - 3- Continued From the data presented here it must be assumed that the highly resistant or- ganisms of the resistant population prefer to absorb the nucleic acid from the normal culture rather than duplicate their own genetic mechanism which is ab- normal as compared with the main population, Table l, TRANSMUT ATIUN OF SULFONAMIDE RESIST ANT &. GObL Nucleoprotein ixtract 3 di none resistant sensitive (mg % 0 +444 +444 +444 2 +++4 +444 +444 5 $++4 +4++4 +444 10 +++ +t+++ ++ 20 +++ +++ - 30 + + ~ = 4 - Continued Table 2, TRANSMUT ATION OF SULFONAMIDE SENSITIVE E. COLT Nucleoprotein Extract Sulfenilamide None_ Resieant = Sensitive 0 ++t+ e+e +444 2 +444 ++++ +++4+ 2 ++ o+e ++ 3 + +++ * § ~ ++ - 7 - + - 10 - zt - Table 3, PLATE COUNTS OF E,. COLI ‘i! SYNTHETIC AGAR CONTAINING VARYING SULFANILAMIDE CONGENTRAT IONS Me 2 Sulfanilamide Straig extract Q 2 2 a2 20 Sensitive none 130 M 2éM 900 0 0 Sensitive resistant 120 M 7M 2 T 100 0 Resistant none 140M 112M 8M 800 T 900 Resistant sensitive 140 M 100 M 420 T 2T 0 Continued TAOLE Ke PREPARATION UF NUCLEIC ACID 1 gram wet cells - .1 M Na citrate in «26 Na desoxycholate, Heat to 50° C, for 10 minutes, Centrifuge, cell debris crude extract discard + 4 volumes of EtOH other precipitate fibrous precipitate and dissolved substance (crude nucleoprotein) di dissolve in M NaCh shake with 1/10 vol amyl aleohol and 1/3 vol chloroform protein nucleic acid