Second interim Eeport 60 the Zatural Sciences Division Rockefeller Foundation May 1, 1956 Project: Genetics of dacteria Submitted byt Joshu: Lederberg Assistent Professor of Genetics University of Wisconsin Madison, wisconsin 1. Gene enzyme relationships in 3. coli, fae atudies of the genetic control of lactase which were initiated during the first year of the project (See Interim Report I, 1949) have been continued. ‘the in- prove! colorimetric methods for determining this enzyme (Rep. I) have resulted in a Clarificstion of the nature of the enzyme adapt..tion illustrated by laet. se. It has Deen well known that HE. coli cells do not attack lactose (or various other sugars besides zlucose) walesse they have been oreviously exposed to the substrite for some period of time. The development of specific enzymatic capacities wader these con- ditions has been called substrete-dependent ensymatic adaptation. The concept that adaptation is bused uoon an actual diversion of protein synthesia towards formation of the adaptive enzyme, uncer the influence of the substrate, is of fundamental im- portance in these stviies, and hus been more cor less generally aeceyied, alveit on father limited evidence. In fact, Deere et. 11, reported, some ten to fifteen years ago, that unadapted cells could be activated by vacuum drying, antisepticsa, or other treatments which altered the permeability of the celle, and concluded therefrom that adaptation involved only a change in ceil permeability which allowed the substrate to gain eecessa to the interior of the eeli. Although the methois used by Doere were rather crude, his observations were nob coutroverted, and poged au uncomforteble dileaus. Usine our direct colorimetric method for lactase assay, we have been able to confirm Deere's observations. However, adepted gelle ere also activ.ted in the satie ratio ae unadapted, so that it can be corcluded that adaptation hae iavolwed a 56 - 100 fold increase in enzyme content. Unndapted cella ordinurtily contnin a Tesidual amount of lactase, not readily detected by the older methods, auounting to 1 + 29 of the fully adapted cells. Activatlon by drying or autolysis causes 2 20 = 39 fold augmentation of the apparent activity, woich would bring unadaptet sells tc the same order of activity as the adanted, intact cells. Unfortunately, certain rather mild treatments, such as standing in K/10 buf- fer, activete cells to a small, but veriable extent, The resulting instabdility ‘ bo ‘ of asssy of assay of intact celle is a troublesome obatacle to kinetic an? other studies on the enzyme in euch cells, and might well be a factor in other stulies of adaptive ensyme formation. fhe concept of a complex genetic control of the adaptation mechanism was men- tioned in the previous report, (Rep, 1). ls concept is supported by the bah:vior of auother mutant recently studied. The lactose analogue, "neolactose." or D- altrose- ~D-gelactoside, is not attacked by wild type 5. coli, when tested by routine bacteriological procedures, Aposrently neolactose is not an affactive atimalus for lactase aieptation, for cells which have been grown on & lactose mediun will attack neolactose. After prolcaged incubstion on neolactese uediun, wild type E. coli celis give rise to mitcnts which will forn lectiuse oa tais me~ dium, and therefore attsek the neclactsse. The matantea, however, have turnel out to be “constitutive” lactase producers; that is, they will produce this ordinarily adaptive enzyme in large amounts whatever the medium on which they are grown. fheir attack of nedlactose ie made possible not by a new adaptive response to tals analogue, but by the fact that the adaptive mechanism has beea disoanse’ with, or probably more accurately, has been triggere? fatrinsinecally by a gonetiz chance. fhe musction has a apecific effect, anc does uot affect the adaptive respousss to any other sugar so far tested includin: galactose. The interaction of tis mutation wit, other genes affecting lectase formation ia under study. The resuits sow, however, that genetic effects may concera any cf a variety of ohaves of anzyme for- mation, and that it 1s impossible with the help of present methods to suecify a gene as the specificity-model or "templite*® for an enzyme. They also show tht the substrate specificities for adeptation and of the enzyme producei may ust be con- gruent, which would ten’ to rule out the hysothesis that adaptation 1s somehow aedicted by the combinition of the substrite with the ame1l amounts of reatduel enzyme usually present in unadapted cells. 2, Gytorenetics of BE. coll K-12. -3e The genetic life cycle of E. coli has revealed a number of complexities not yet thoroughly unravelled, At present, a mass of detailed information has been collected, which cannot well be preaented until some generalisations have been deduced from it. Fhe diploid phase seema to undergo three types of aberrant be~ havior: (1) elimination, so that the "“diploldis® becore hemizygous or nonseenic for certain blocks of genes, and (2) "double reduction,” so that some blocks of «anes, originally heterozygous, become homozygous, and (3) probably as a sonsecusace of the eliminations, non-random segregations, viz., thet alternative alleles do not occur with equal frecusacy amons tha segregents. Although the situation has noi yet been Clarified, it appears lixely that the ~berraiions can be best Lluterpretec in toras of s chromcsomal system like thst of hisher forms, Gytologicel studies auve been initiated with the alm of comparing ani distin- quigshing the nuclear morphology of haploid and diploid cells of EB, coli. the nuclear components of thase tyoes of cells cnn be distinguished: the disioic cells generully show a more disperse, larger aggregate of nuclear material, containing a larger mumber of resolvable granules in each geregate, but with the individual gvanules amaller than in the haploid. However, we are not yet able to interpret this consistant difference in appearcace in terma of their veastic structure. 3. Genetie aspects of becterteide. It has often been speculated, without proof, that the lethal effects of ra@istione and other chemical and physical szgents on bucterts might have « gzeastie basis, euch as induced lethal mutations, The divleit eultures of BE. coli now at our disposal permit an examination of this question. A number of agents huve been found which have profounl genetic disturbunces correlated with bactericidal «ction, Wat the disturbences are mostly in the fory of haploidizetion, i.e., the renoval of en entire gene set, rather than e lethal mutation (the equivalent of removal of a single gene). This would euggest thet the lethal aspects of these agents are more parallel to the so-calied physiological or Karypathological effacts of radiations on nuclei of higher forms, than to the specific structural (stide-7) effects on chromosomes which have enezged the major interest of geneticists. the nature of the haploidization has not yet been thoroughly analysed, but = tubwl: tion of the reagents which do, and do not, resemble radiations in their paetericidal effects may be of interast to the gencrel vroblem of bictericide. - Heploidizing bactericiies Non-haploidizing bactericices Ultraviolet light* Heat X-rays * Methyl green Nitrogen mustard * Pyronine Y ** Forng] dehyde * Streptomycin Hydrogen peroxide* Todine Dimethyl sulfate Todoacetimide Acetic anhydride Ethyl carbamite** * Jeli-esteblished as mtsgenic chemicals (for organisms other than b.cteariz). “*CLoined aa sossible mut-zens for bacterie by other worxers. It may be noted that a common property of the agents listed as haploidizine is their potentiality for inducing substitutione on amino and other reuctive erouss of vreteine or nucleic acids. 4, Seleetion of genetic recombinants with bacterial growth inhibitors. In vlace of a nutritional selection of wild-type recomolnente from s mirture te £ Atatinet biochemical mut-nts, it is fessible to select dually resistant racon- binsntsa of B, aoli from a mixture of cultures each resistant to a distinct .nti- Rogbert.’ gomsound, such as streptomycin ond solium azide. This technicue is » & useful euxilisry to the nutritional methed for ©. coli, and should make sossible tests for recombination in organiams (like the vathogenic cocel) net reudily aneneble to nutritional methods. PUBLICATIONS fhe folloving publications pertain to this project, and have not yet been cited, 1. 2. 3. 2. Aberrant heterozygotee in Zwehcrichia coli. Pree. Bat. Acad. Sei. U.S., 353 178-84 (1949). Mrect utilization of maltose by Escherichiz coli. J. Biol. Chem. 179:921~34 (1949). Bacterial variation, Ann, Rev. Microbiol. 321-22 (1949). fhe selection of genetic recombinations with bacterial growth inhibitors, J, Baet, 59:211-215 (1950). Tu preparation: The ~-D-galactoeldase of Escherichia coli K-12, Genetic aspects of bacteriaide. Physiology of the gene: genetic studies on becterila In press? foe isolation and oharacterization of biochemical mutants of bacteria. (Methods of Nedicel Resexrch, Folume III, 1950). Inheritance, Variation and Adaptation. Chapter in Bacterial Physiology. Wilson and Werkman (Bds,).