c Indian Academy of Sciences

SPECIAL FEATURE

Joshua Lederberg – a remembrance

ABHIJIT A. SARDESAI* and J. GOWRISHANKAR*

Laboratory of Bacterial Genetics, Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad 500 076, India

Joshua Lederberg, an iconic figure in , passed It was the subsequent work of William Hayes and of away on 2 February 2008 at the age of 83. Many obituar- Lederberg which led to delineation of the underlying mech- ies have since appeared describing his work in and outside anism, namely discovery of the fertility or sex factor (the F- of science (Balaram 2008; Blumberg 2008; Crow 2008a,b; ), mediating conjugative gene transfer, and the word Oransky 2008; Sgaramella 2008). As a practising micro- conjugation gradually came to be applied to the phenomenon biologists, we take retrospective glimpses in this article at (Cavalli et al. 1953;Hayes 1953; reviewed in Firth et al. some of his research contributions that have profoundly in- 1996). It is now recognized that the gene transfer detected fluenced the development and growth of prokaryotic biology. by Lederberg and Tatum was tantamount to their winning For more personal accounts of his career, including specific the jackpot (in more ways than one, see below), since the examples of his legendary intellect, readers may refer to the bacterium E. coli K12 fortuitously chosen by them for their two articles cited above by James F. Crow, a close colleague work differs from the majority of other enterobacteria in that of Lederberg, and the one by Baruch S. Blumberg, a long it carries a natural mutation in the gene encoding an inhibitor time associate. of fertility factor, and also allows for Hfr formation that is Lederberg’s discovery of in the mid essential for conjugative mobilization of the chromosome 1940s is perhaps his most celebrated contribution. In hind- (Miller 1992). The biology of the F-plasmid is now under- sight it would be fair to say that the early experiments by stood in great detail and the Lederberg and Tatum experiment Lederberg carried out in the laboratory of in was the cornerstone for exploiting the use of Hfrs (high fre- pursuit of his Ph.D. clearly fall in the category of shots in the quency recombination strains) in establishing that the chro- dark whose outcome was not certain. However, Lederberg mosome of E. coli is circular and in the development of its had built into the experiment a selection, a sort of a barrier. genetic map. Fast-forward to recent times and conjugation is It was reasoned that progeny arising from the process of ex- now considered as a mainstay mechanism that mediates hor- change of genetic material during coculture of two geneti- izontal gene transfer (HGT), a process thought to contribute cally distinct bacterial cell types would perhaps be the only to microbial diversity in nature (Thomas and Nielsen 2005; ones to overcome the barrier (a medium permitting growth Babi´c et al. 2008). only of recombinants) and hence be isolated, which was Following on from his discovery of bacterial conjugation, the case (Lederberg and Tatum 1946; Tatum and Lederberg Lederberg entered into the most productive phase of his sci- 1947). As an aside, it is worth noting that in these, their entific career. With Norton Zinder, Lederberg came upon two earliest papers, the word “conjugation” is not explicitly the process of -mediated generalized transduc- mentioned; rather the process is plainly referred to as gene tion (Zinder and Lederberg 1952). The Zinder–Lederberg recombination in . The reason for describing paper comes out as a rather interesting read. With custom- this phenomenon so literally (and a good example for other ary rigour, the authors go to great lengths to demonstrate mortals to follow) is apparent: their experiments did not il- that the activity of an entity (named FA: filterable agent) luminate the mechanism that led to the exchange of genetic mediating genetic exchange is similar to that of a bacterio- material. phage. Perhaps one sentence from the Zinder–Lederberg paper sums up the phenomenon—“that the activity of FA parallels the characteristics of the donor cell”. In effect, Zinder and Lederberg were describing the phenomenon of *For correspondence. E-mail: [email protected]; [email protected].

Keywords. bacterial genetics; conjugation; ; bacteriophage; mapping; F-plasmid; lysogeny.

Journal of Genetics, Vol. 87, No. 3, December 2008 311 Abhijit A. Sardesai and J. Gowrishankar phage P22-mediated generalized transduction in Salmonella selection rather than being ‘directed’ by the selection. Note typhimurium (now known as S. enterica). Coming to grips that this concept did exist at the time, Lederberg’s work be- with the principles of phage-mediated generalized transduc- ing preceded by that of both Luria and Delbr¨uck (1943) and tion and employing them in a suitable manner has constituted Newcombe (1949). It was just that the replica plating tech- the A, B, C of microbial genetics ever since. nique allowed a neat, more direct and visual representation A few years prior to the discovery of generalized trans- of the Luria and Delbruck model. duction, Esther Lederberg (Lederberg’s first wife) identified In this remembrance of the scientific career of Joshua the temperate bacteriophage lambda, and along with Joshua Lederberg, we have tried to bring out snippets of his fa- Lederberg was instrumental in the early elucidation of phage mous work that has left behind legacies for the disciplines lambda biology. Two pieces of their work merit mention. of molecular biology in general and bacterial genetics in par- First, they were able to show that phage lambda, apart from ticular, and the following words from Kohiyama et al. (2003) existing in a conventional phage-like lytic stage, could also perhaps provide a true measure of the impact of Lederberg’s exist as a lysogen integrated in the genome as a neutral en- discoveries: “It is difficult to overstate the importance of the tity that behaves as a genetic locus (Lederberg and Lederberg discovery of bacterial sex for the development of molecular 1953). In the same study they obtained evidence that the genetics and biology. Even the ring structure of the bacterial lysogenic state of lambda lay in close association with the chromosome was first revealed by the study of conjugation. neighboring genes for galactose fermentation (gal) and with Conjugation, transduction and transformation are still basic stunning accuracy postulated the existence of a site on the tools for cloning, characterizing and sequencing all genes bacterial chromosome to which ‘lambda remained bound’. and genomes”. To this we may add that experiments involv- That site is now referred to as the phage attachment site. In a ing conjugation also contributed directly to discovery of the follow up to their efforts in elucidation of biology of lambda, mechanisms of homologous recombination in bacteria (Clark Esther and Joshua Lederberg described the phenomenon of and Margulies 1965; Emmerson and Howard-Flanders 1967; specialized transduction (Morse et al. 1956), involving the Willetts and Mount 1969). isolation of λgal as a product of imprecise excision of a lyso- For his work, which laid the foundations for the growth gen following illegitimate recombination between sequences of molecular genetics of phage and bacteria as a discipline, on lambda and those on the adjacent bacterial DNA. The Lederberg along with Edward Tatum and did λgal phage particles carry the genes of the galactose operon, win the jackpot of the Nobel Prize in physiology or medicine located on one side of and in the immediate vicinity of the in 1958. As is apparent from obituaries of Lederberg written lambda , and could mediate the transfer of the gal by his close associates, later on his research interests broad- genes. This was possibly a forerunner for the first genetic ened considerably in areas such as exobiology and artificial engineering experiment, and in today’s context one could say intelligence, and his ideas and views on the practice of sci- that the gal genes had been cloned (on a ). ence had a deep influence on science policy in general in the To suggest that these contributions of Lederberg were USA and around the world. All through his life, Lederberg pivotal in the growth and development of the field of phage maintained a passionate interest and commitment to science. and bacterial molecular biology would rank as an understate- He was also a member of the editorial and advisory board of ment. It might interest the reader to know that λgal phage this journal from the time that its publication was restarted served as a critical resource for validating one aspect of by the Academy in 1985 until his death. the famous ‘Campbell’ model of site-specific recombination Rewind to conjugation. This technique has recently been (Campbell 1961). λgal phage was used for determining the employed independently by two groups to perform large- DNA sequence junction between a lysogen and the adjoin- scale synthetic-lethal interaction studies in E. coli (Butland ing the bacterial DNA, thus providing persuasive evidence et al. 2008; Typas et al. 2008). Furthermore, a recent issue that the prophage ends in a lysogen are covalently linked to of Science carries a paper by Babi´c et al. (2008) entitled “Di- the adjoining bacterial DNA following integration (Campbell rect visualization of horizontal gene transfer”, in which the 1993; Landy and Ross 1977). Note that a precisely excised authors describe an experimental system to visualize, by flu- lambda phage will not bear adjoining bacterial DNA. oresecence microscopy, the DNA actually transferred during No description of Lederberg’s research would be com- bacterial conjugation from donor to recipient cells, in real plete without mentioning the famous replica plating tech- time. The novel features of conjugation that have been high- nique he devised (Lederberg and Lederberg 1952). It is lighted by this work are (i) single recipient cells could re- pleasingly amusing in hindsight to read the opening sentence ceive DNA more than once and at different times, (ii) that of the paper: “A frequent chore in bacteriological work is the F-pilus serves not only to establish contact between mat- the transfer of isolates from one substrate to other selective ing cells but also as a channel for DNA tranfer over distances or indicator agar media.” Microbiologists across generations as large as 12 microns separating donor and recipient (each would attest to this. Replica plating has been widely used of which is only 1 to 2 µ in length), and (iii) that the trans- since, but the Lederbergs employed it to reconfirm an impor- ferred DNA is subject to two competing processes, namely tant concept, that mutations (in bacteria) arise in absence of degradation by the RecBCD nuclease and homologous re-

312 Journal of Genetics, Vol. 87, No. 3, December 2008 Joshua Lederberg (1925–2008) combination by RecA, with the latter predominating in 97% Emmerson P. T. and Howard-Flanders P. 1967 Cotransduction with of the cells. Furthermore, the authors demonstrate that E. thy of a gene required for genetic recombination in Escherichia coli cells undergo 1.4 sister chromatid exchanges on average coli. J. Bacteriol. 93, 1729–1731. per cell division. Firth N., Ippen-Ihler K. and Skurray R. A. 1996 Structure and function of the F factor and mechanism of conjugation. In Es- One cannot but feel a sense of heavy irony at the coinci- cherichia coli and Salmonella: cellular and molecular biology, dence in the timing of the publication by Babi´c et al. (2008). 2nd edition (ed. F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, Thus, the biological process of conjugation that Lederberg E. C. C. Lin, K. B. Low B. Magasanik et al.), pp. 2377–2401. had initially inferred and conceptualised almost 60 years ago, American Society for Microbiology, Washington. has now been visualized in real time and in single cells of- Hayes W. 1953 Observations on a transmissible agent determining sexual differentiation in Bacterium coli. J. Gen. Microbiol. 8, 72– fering perhaps a fitting tribute to his passing. Silhavy and 88. Gitai (2008) record a similar sentiment in their News and Kohiyama M., Hiraga S., Matic I. and Radman M. 2003 Bacterial Views feature covering the synthetic-lethal interaction stud- sex: playing voyeurs 50 years later. Science 301, 802–803. ies, when they write: “Joshua Lederberg passed away this Landy A. and Ross W. 1977 Viral integration and exci- year. We wish he was still with us to witness how his Nobel sion.structures of the lambda att sites. Science 197, 1147–1160. Prize-winning discovery opened the door for comprehensive Lederberg E. M. and Lederberg J. 1953 Genetic studies of lyso- analysis of genetic interactions in E. coli”. genicity in Escherichia coli. Genetics 38, 51–64. Lederberg J. and Lederberg E. M. 1952 Replica plating and indirect selection of bacterial mutants. J. Bacteriol. 63, 399–406. Lederberg J. and Tatum E. L. 1946 Gene recombination in Es- Acknowledgements cherichia coli. Nature 158, 558. LuriaS.E.andDelbr¨uck M. 1943 Mutations of bacteria from virus Work in the laboratory of Bacterial Genetics is supported by the sensitivity to virus resistance. Genetics 28, 491–511. award of a Centre of Excellence project in Microbial Biology by Miller J. H. 1992 A short course in bacterial genetics: a laboratory the Department of Biotechnology, Government of India. manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New References York. Morse M. L., Lederberg E. M. and Lederberg J. 1956 Transduction Babi´c A., Lindner B. L., Vuli´cM.,StewartE.J.andRadmanM. in Escherichia coli K-12. Genetics 41, 142–156. 2008 Direct visualization of horizontal gene transfer. Science Newcombe H. B. 1949 Origin of bacterial variants. Nature 164, 319, 1533–1536. 150–151. Balaram P. 2008 Scientists, teachers and writers: remembering a Oransky I. 2008 Joshua Lederberg. Lancet 371, 720. remarkable trio. Curr. Sci. 94, 551–552. Sgaramella V. 2008 Joshua Lederberg (1925–2008). DNA Repair 7, Blumberg B. S. 2008 Joshua Lederberg (1925-2008). Nature 452, 1004–1005. 422. Silhavy T. J. and Gitai Z. 2008 Sex to the rescue. Nat. Meth. 5, 759– Butland G., Babu M., Diaz-Mejia J. J., Bohdana F., Phanse S., Gold 760. B. et al. 2008 eSGA: E. coli synthetic genetic array analysis. Nat. Tatum E. L. and Lederberg J. 1947 Gene recombination in the bac- Meth. 5, 789–795. terium Escherichia coli. J. Bacteriol. 53, 673–684. Campbell A. M. 1961 Episomes. Adv. Genet. 11, 101–145. Thomas C. M. and Nielsen K. M. 2005 Mechanisms of, and barri- Campbell A. M. 1993 Thirty years ago in genetics: prophage inser- ers to, horizontal gene transfer between bacteria. Nat. Rev. Mi- tion into bacterial chromosomes. Genetics 133, 433–438. crobiol. 3, 711–721. Cavalli L. L., Lederberg J. and Lederberg E. M. 1953 An infective Typas A., Nichols R. J., Siegele D. A., Shales M., Collins S. R., Lim factor controlling sex compatibility in Bacterium coli. J. Gen. B. et al. 2008 High-throughput, quantitative analyses of genetic Microbiol. 8, 89–103. interactions in E. coli. Nat. Meth. 5, 781–787. Clark A. J. and Margulies A. D. 1965 Isolation and characteri- Willetts N. S. and Mount D. W. 1969 Genetic analysis of zation of recombination-deficient mutants of Escherichia coli. recombination-deficient mutants of Escherichia coli K-12 car- Proc. Natl. Acad. Sci. USA 53, 451–459. rying rec mutations co-transducible with thyA. J. Bacteriol. 100, Crow J. F. 2008a Joshua Lederberg, 1925–2008: A tribute. Genetics 923–934. 178, 1139–1140. Zinder N. D. and Lederberg J. 1952 Genetic exchange in Crow J. F. 2008b Joshua Lederberg 1925–2008. Nat. Genet. 40, 486. Salmonella. J. Bacteriol. 64, 679–699.

Received 20 October 2008; accepted 20 October 2008 Published on the Web: 11 November 2008

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