LASKER~KOSHLAND SPECIAL ACHIEVEMENT IN MEDICAL SCIENCE AWARD COMMENTARY I never met a microbe I didn’t like Stanley Falkow At the age of 11, I read Paul de Kruif’s Microbe Hunters, which dramatized the discovery of bacteria and viruses and their roles in human disease. The heroes of Microbe Hunters—Louis Pasteur, Robert Koch and others—became my heroes, and I dreamed of becoming a bacte- riologist, doing research on the bacteria that cause disease. I was lucky enough to fulfill my boyhood dream; however, I could never have imagined the path I eventually followed, or how much my views of microbes and disease would change (and continue to do so) in the process. Of course, I did not make this journey alone. During the five decades I worked as an active scientist, I helped train over 100 graduate Figure 1 Lab alumni reunion in 2004, Falkow/Tompkins home, Hamilton, Montana, USA. Photo students, postdoctoral fellows and clinical fel- courtesy of Manuel Amieva. lows, and collaborated with 75 other scientists (Fig. 1). Each of us, in our own way, wondered, Professor Herman Chase, a mouse geneticist, nicity. I was unable to transfer any gene from “What is a pathogen?” thought about my question and announced Salmonella or Shigella that altered the measur- that he had just the book to start me on my able pathogenicity or host range of another Entering the genetic and molecular world voyage, the just-published compilation The Salmonella species, or that made E. coli K-12 I was a hospital bacteriologist and an autopsy Chemical Basis of Heredity1. In this volume, I pathogenic2,3. diener before I became a graduate student. learned for the first time about the structure When I presented these results at a Cold Thus, I learned about the world of microbes of DNA and embarked on my research during Spring Harbor meeting in 1963, the opinion from a practical standpoint before I learned the the beginning of what Salvatore Luria called was almost unanimous that I was wasting my tools to perform research. The medical bacte- the “Golden Age of Molecular Biology.” At the time and ought to be concentrating on more riology of the 1950s focused on differentiat- time, bacterial genetics was basically restricted important biological questions. Indeed, at ing the ‘good guys’ from the ‘bad guys’, and a to Escherichia coli K-12. that time there was a growing consensus that pathogen was simply defined as any organism Shortly after I became a student, Lou Baron infectious diseases were no longer of interest that caused disease. In basic bacteriology I was at the Walter Reed Army Institute of Research for Western society. So, at age 30, I put my taught that bacteria were Schizomycetes— described for the first time the transfer of dream of doing research on the meaning of ‘asexual primitive plants’. So, it was hard to genetic information from E. coli to Salmonella bacterial pathogenicity to rest for a time and think of them as being inherently virulent. typhi. I was anxious to use the tools of genet- devoted my energy to examining the molecu- When I entered Brown University as a grad- ics and molecular biology to establish the spe- lar nature of extrachromosomal elements— uate student in 1957, I pestered my professors cific genes that defined the difference between now called plasmids—with help from Julius asking what they thought made pathogens pathogens and non-pathogens. There were, Marmur (then at Brandeis University)4. I different from non-pathogens. Professor C. A. after all, E. coli that were clearly part of the focused especially on R plasmids, as these (‘Doc’) Stuart encouraged me to learn genet- normal flora and E. coli that caused diarrhea mediators of antibiotic resistance were of ics as a foundation for answering the question. in infants. So, I immediately contacted Baron, clear medical significance5. who supplied me with the necessary cultures Stanley Falkow is the Robert W. and Vivian K. to study this problem. Plasmids and pathogenicity Cahill Professor of Microbiology and Immunology I performed conjugation experiments using I discovered the joys of teaching in 1967 and and of Medicine, Department of Microbiology E. coli K-12 donors and clinical isolates of moved to Georgetown University. The follow- and Immunology, Stanford University School of E. coli and Salmonella. I was excited when I ing year I met H. Williams Smith at a meeting Medicine, 299 Campus Drive, D039, Stanford, could actually detect E. coli surface antigens in London. He was a veterinarian who had used California 94305-5124, USA. in Salmonella. However, my conjugation basic bacterial mating experiments to show e-mail: [email protected] experiments revealed little about pathoge- that diarrhea in pigs and calves depended on NATURE MEDICINE VOLUME 14 | NUMBER 10 | OCTOBER 2008 xxvii COMMENTARY graduate students to Herb’s lab to pursue the idea. It worked, and we cloned the first virulence determinant of bacteria—the E. coli heat-stable enterotoxin—in my laboratory10. The implications of recombinant DNA tech- nology were enormous, of course. Because of my training in medical microbiology, their impact on me was not just scientific. Indeed, I participated in the historic Asilomar meeting on the societal impact of recombinant DNA. I served on the first I served on the first US National Institutes of Health Recombinant DNA Advisory Committee, established in 1974 in response to public concerns regarding the public health and safety issues of manipu- lating genetic material using recombinant DNA techniques and the potential ethical and social implications of the research. The committee was initially charged with draft- ing guidelines governing the safe conduct of recombinant DNA research by outlining appropriate biosafety practices and contain- ment measures. These guidelines, now known Figure 2 Photo taken on 25 May 1967 during my lecture at the Symposium on Infectious Multiple Drug as the NIH Guidelines for Research Involving Resistance, held at the Georgetown University School of Medicine with support from the US Food and Recombinant DNA Molecules, were first pub- Drug Administration. In the front row are, left to right, Arthur K. Saz (lighting his pipe), Piet A. Guinée, lished in 1976 and have evolved over time to Naomi Datta, David H. Smith (a Lasker Award Winner) and Tsutomu (Tom) Watanabe. The last four were include other aspects of gene manipulation, instrumental in discovering R plasmids and demonstrating their significance in clinical medicine. including genetic therapy. This was a time- E. coli strains that possessed two plasmids, one likely that these extrachromosomal elements consuming and difficult task, and it was not encoding one or two enterotoxins, and a sec- were an important part of bacterial evolution, ond encoding an adherence factor that specifi- including the evolution of pathogenicity. cally recognized the epithelial cells of the small bowel6. Willie asked me if my students and I Recombinant DNA, gene transposition could use our molecular methods to examine and a return to understanding these virulence genes. By now we had become pathogenicity adept at isolating plasmids from bacteria on In June 1972 I moved to the University of the basis of their circularity, and I was actively Washington in Seattle, where I would have a engaged in using DNA hybridization to exam- more active role in teaching medical micro- ine the relationships among plasmids. biology and directly participating in research Smith’s work became the foundation on infectious diseases. Soon after moving, I for looking at whether certain E. coli from attended a joint meeting of US and Japanese humans possessed a similar plasmid arsenal plasmid researchers in Hawaii (Fig. 3). and caused traveler’s diarrhea. Our laboratory A major focus of the meeting was to gain showed that the plasmids encoding entero- an understanding of how R plasmids acquired toxins from pigs had closely related counter- resistance genes and whether R plasmids were parts in humans. Indeed, they were related to natural co-integrates of distinct replicons. A the classic F factor of Joshua Lederberg and concurrence of results on the origin of R plas- to certain R plasmids7. Working with Naomi mid resistance genes discussed one evening in Datta and Bob Hedges, we discovered that the unlikely setting of a Waikiki kosher deli- there were many distinct groups of R plasmids catessen led to the idea of joining and splicing (Fig. 2)8. Among some of them, the antibiotic- DNA using restriction enzymes. My only con- resistance genes seemed to have a common tribution was as a witness, occasional commen- source, but the replication machinery, the tator and donor of replicon RSF1010, known restriction-modification loci, and the pro- to have a single EcoR1 cleavage site, for the Figure 3 The US–Japan plasmid meeting in teins that permit transmission of DNA from first pilot cloning experiments performed by Honolulu, Hawaii, November 1972. Close-up a donor to a recipient were different. Yet Stanley Cohen, Herb Boyer and their co-work- from a picture taken the day after the meeting plasmids from a single group could carry ers9. However, I was aware of the implications in the kosher deli, where we discussed the experiment that led to gene splicing. Left to right: antibiotic resistance or have one or more of the work. Indeed, I extracted from Herb Stanley Falkow, Robert H. Rownd, Herbert W. enterotoxin or adherence genes. It was as if and Stan the promise that, if the experiment Boyer, Stanley N. Cohen, Toshihiko Arai, Charles different gene cassettes could be inserted or succeeded and gene isolation and amplifica- C. Brinton Jr., Richard P. Novick (partly hidden) taken out of the same plasmid. It seemed tion became a reality, I would send one of my and an unidentified Japanese scientist.