Q36 Research in Microbial Genetics, 197-1957 Department of Genetics, University of Wisconsin Professor J, Lederberg A, Main lines of research, 1, BACTERIAL SEXUALITY. The discovery of sexual reproduction in Escherichia coli is the starting point of a direction of microbiological research, which bas helped to restore the bacteria to a definite place in the scheme of terrestrial life. The mating process, originally inferred from cross-breeding experiments, has been observed under the microscepe and consists of a esli~ to-cell pairing or conjugation, during which genetic material passes from one partner to the other, An elaborate system of mating types controls which combirations sre sexually compatible, © 2, RECOMBINATION ANALYSIS. This system ef crosa@breeding allows a detailed aralysis of the genetic control of specific physielogical processes, For example, the ability te form an enzyme, lactase, is controlled by a consicere able number of different genetic loci, some of which have been found to Ine fluences different aspects of enzyme synthesis, Some of these lcci are found in very compact clusters and they interact in such a way ss to indicate regional organization of the bacterial chromosome in relation to the physio- logical actions of thes genes. Mutations for drug resistance are likewise found to relate to changes of specific loci, 3, LYSOGENICITY, It has been known for many years that certain bacterial strains might harbor viruses ina latent form, a relationship called ‘lyso- genicity', Recombinatienal. analysis of lysogenic strains of 5, coli haz shown the intimacy of the relationship between the provirus and the bacteris] host, within whose chromosome it occupies a definite site, This finding has bolstered the speculation that a virus consists of a potentially autonomous segment of the host's chromosome-«the generality of this statement can, of course, be questioned, Q36 lh, TRANSDUCTION. This is another mechanism ef recombination, whereby smali frapments of tie heredity of ome strain can co tranemitoed bo another, in Salmonella, these fragments are carried by phage, being incorporated more or less adventitiously during the growth of the virus on the first host. In addition to its intrinsic interest as an unsuspected festure of virus- cell interactions, transduction also affords another tool for genetic analy- sis. In E, coli, another type of transduction has been found in which the fragment is directly linked to the provirus. The interaction between the input fragment and the chromosome of the recipient celis has heen studied in detail, and found to parallel the events of crossingeover in hybrid zyzotes which have received a full complement from both parents, 5. IMMUNOGENETICS OF SALMONELLA, The pattern of naturally occurring anti- genie combinations in these bacteria has been a long-standing puzzle of considerable theoretical and epidemiological interest, By transductional techniques, it has been possible to reconstruct new types, and to explain the existing types as the recombinations of various antigenic factors, In addition, the phenomanon of phase variation has been clarified as an alter» nation in the local functional "state" of an antigen-determining gene, (This dimension of genic variation can be contrasted with mtational changes of specificity, and offers new grist for the model-building mills of theoretical embryology). 6, ABORTIVE TRANSDUCTION AND LINEAR INHPRITANCE, Another consequ nee of transduction, besides the developzent of genetically stable recombinant types, is a clone in which the new trait is exhibited by only one or a few celis, When these cells divide, the trait is passed on to only one of the progeny, thus creating a single line of descent, This pattern of linear inheritance (which also has some developmental analogies in stem-cells) can be explained by the transmission of a nonereplicating gene-product, or perhaps a damaged gene itself, Q36 7. PHYSIOLOGICAL GENETICS OF GALACTOSE ENZYMES: POSITION 2Prict, Prarncve tion in E, coli by the phage lambda uniquely concerns a group of genes affecting the metabolism of galactose, Kalckar and Kurahashi have shown that a number of these mrtants are deficient either in a) the enzyme galactokinase, or b) the enzyme uridine~di-phosphate-glucose transferase, (These mutants have precisely the same biochemical defect as humans suffering from the hereditary disease, "galactesemia," } By transductional methods, we have found that the mutants within group a) show a cis-trans position effect, that is hetero» genotic sells which have the constitution +e/et+ are umable to utilize galactose, while cells of the constitution ++/+— can, The same holds for tte watants in croup b. However, although both groups of mutants are closeir linked to each other, a mutant from a) does not show the position effect with a mutant from b). That is, the +./.+ combination here will produce both enzymes, These results are explicit suppert for a unique, coherent functions] segment cf a chromosome concerned with a single enayme, However, there are some apparent exceptions to the rule which may ceive still further insight inte these relationships, 8. ORIGIN OF DRUG@RESISTANT STRAINS OF BACTERIA. There has been mach con~ troversy over this question, some authorities holding that antibacterial chemicais might react directly with the hereditary framework of the bacteria to produce resiatant, mutants; on the other hand, much indirect evidence pointed to the sporadic cecurrencte of such mutants, independently of the drug, whose function is simply to provoke the selective ovtgrowth of the rare re- sistant forms, By various technical innovations, it became possible to iso. late resistant mitant clones without ever exposing them directly to the drug. That spontaneous mutation can account for at least some examples of drug resistance is therefore now beyond question, though there remains some dis- cussion as to the possible operation of the alternative mechanism in a few Q36 unsettled cases. ©, PROTOPLASTS, L«PORMS, ANDO TRE MECHANISI CF ACQION OF Vi OTan ay, puzzling observations (some quite ancient) on atypical growth forms of bacteria have been coordinated into a simple working hypothesis, based on observations on the action of penicillin on E, coli, The welleknown fact thet penicillin is lethal only to growing bacteria, which then lyse, has been the basis of the very useful 'penicillin-method! for the isolation of growth-factor depemlent miants. Recently, it was found that this lysis could be forestalled by maintaining the treated cells in a medium containing hypertonic levels of sucrose (or certain other solutes), Instead of lysing, the treated cells expand, and the intracellular protoplast shades the Limiting cell wall, becoming a spherical globule, These protoplasts remain viable se long ae they are kept in the sucrose, and will regenerate walls to revert to normal bacillary shape if the penicillin is removed, If they are placed in water or ordiniry dilute medium, however, the protoplasts lyse and, of course, lose their viability as a result. Therefore, the bactericidal aetion of penicillin may be explained as an inhibition of cellewall synthesis, virile protoplasmic synthesis contimes until the wall has burst, | In penicilline-sucrose broth, the protoplasts increase in substance, bus fall to multiply, simply becoming larger and larger spheres, In agar zediun, however, the expanding protoplast is confined by the agar meshwork, and therefore forms blebs and processes at the points of least resistence, These biebs enlarge and eventually round up, the reiteration of the process piving a colony of protoplasts of various sizes: this is, in fact, the Letype groutn of previcus authors. As a rule, the proteplastic colonies resume baciliary form, even after many serial passages, when planted into medium without penicillin, However, from time to time, mitations have occurred which imposed a genetic bleck to some element of cellewall synthesis, for example in the biosynthesis of diaminopimelic acid, Thess mutants correspond to the Q36 genetically stable 'Leforms? of other workers, The subject has formerly been dominated by Life-cyclic ov adaptive, rather than mechanistic hypotheses, These protoplasts have become useful objects of biochemical study, e.g. of protein and nucleic acid synthesis, in the hands of other workers. My" own interest in the problem was motivated in part by the hope that wall-less protoplasts would be more receptive to the penetration of genetically active mucleic acids, This hope is still unfulfilled, but remains the subject of -contimied experiment. Salecued References wwe WTA AAA BRERA UE RAS MN eng SA TATA, § Problems in wlerobial genctics, Lederberg, Ju, B. M. Lederberg, No D, Zinder and E, R, Lively. 951 Recombination analysis of bacterial heredity. Cold Spring Harber Symp. L6shi39hh3., 1951, Lederberg, J./ Genetic studies with bacteria. In: Genetics in the 2040 Century, MacMillan, New York pp. 263-289, Lederberg, J, and E. HM. Lederberg, 1952 Replice plating and indirest selection of bacterisl mtants, J, Bact, 631399):06, Zinder, No D. amd J, Lederberg, 1952 Genetic oxchange in Selmeorelia, J, Bact, 6h, 2679065 O95 Lederberg, Jo, L,. Cavalli-Sforza and E. M. Lederberg, 1952 Sex compati- bility in Escherichis coli. Genetics 372720-7320. Lederberg, E. M, and J. Laderherr. 1953 Genetic studies of lysosuninity tn on ‘ &2 ay te Escherichia coli. Cenoties 30: vy . Cavalli-Sforgs, LL. L, amd Jd. Lederberg, 1953 Genetier of ros bacterdel inhibitors Symp. on groubh inhibition and che International, Congress of Mier. sbiology, | Rowe, po. LOS 1)7, Lederberg, J. 1955 “es ombination mechaniems in bacterds, Jour. Celi, Comp, Physiol,, Suppl. 2 (75-107). Symp. on genetic rece: abinatic Ty, ORNE. tesenborgy J, and &, M, Lederberg. 1956 Infeetion and heredity, Chap. 5, 1Gi-19), in Cellular mechanisms in differentiation ard growth. (Lith OPI Soc, Growth and Development; D, Rudnick, Ed.) Princeton Univ, Press. Bach, 732ibh. ¢ Lederberg, ¢. 1957 Mechanism of action of penicillin. d : Lederberg, J. 2986 Genetic transduction, Amer, Sed. bhs6h 280. marae &, 1956 Conjugal pairing in Escherichia coli. J. Bact, The Lg FohF6. Ledarberg, J. and 7. Timo, 1956 Phase variation in Salnenela, Geretics bh: 736757, Morse, Mo La, E. M. lederberg and J, Lederberg. 1956 Transduebional heise genotes in dgcherichia coli, Cenetics Mls: 37582775 » Lederberg, J. 1956 Linear inheritence in transductional clones. Genetics 41:845~-871. 936 C, As important e contribution as findings of fact je the traiming of graduate students and research fellows, Some of those, who have worked in the laboratory, and are now pursuing their own research programs in microbial genetics are: Dr, N.D, Zinder Rockefeller Institute fer Medical Research New York Dr, Mab. Morse Depts Biophysics, U. of Colo. Medical Center Denver © Dr. SG. Bradley Dept. Bacteriology, U. of Ninn, Medical School Minneapoli Dr, P.D, Skaar Dept. Geneties Carnegie Institution Cold Spring Harbor N.Y. Dr. Brace Stecker Lister Institute for Preventive Medicine Lonion Dr, Aleck Eernstein Central Public Health Laboratory London Br, L. Ly, Cavalli-Sforza Serotherapeutic Institute Milan Professor Sydrey D. Rubbe Dept, Microbiolecy, University of Melbourn: