An Interview with Manuel F. Varela and Ann F. Varela: Esther Zimmer Lederberg

Oct 31, 2021 by

Esther Miriam Zimmer Lederberg

Michael F. Shaughnessy

So many of nature’s most beautiful creations are very small and often overlooked.”

—Esther Lederberg

1) Esther Lederberg was the first to isolate λ bacteriophage. We almost have to start with this statement and work around her life. Why was this event important in the realm of science and bacteria?

Indeed, Dr. Esther Miriam Zimmer Lederberg was the first human being in recorded history to have provided evidence for the isolation of bacteriophage lambda (λ), one of the most famous viruses in the history of molecular biology. Lederberg went on to make significant discoveries dealing with lysogeny, conjugation, and genetic recombination.

The discovery by Lederberg of phage λ has been described in scientific lore as the result of a laboratory accident. In 1951, Lederberg, a graduate student, had reported in an abstract that she had isolated a mutant strain of an Escherichia coli, called W-518, using UV light. The W-518 strain of bacteria was sensitive to infection and lysis by bacteriophages. The mutant Escherichia coli W-518 could, thus, be used as an indicator strain of phage infection. Lederberg studied the indicator strains that are sensitive to phage infection and capable of lysis, permitting themselves to be killed by the phage virus. The lysogen, the bacterial host, was considered by Lederberg as a living phage carrier.

In the groundbreaking experiment, the “accident,” Lederberg mixed her mutant Escherichia coli indicator strain W-518 with the original parental strain, called Escherichia coli K-12, which was known to be lysogenic, i.e., capable of converting to a lytic state. Out of this mixed culturing experiment, where she combined mutant and parent bacteria in the same test tube, Lederberg found novel signs of the λ phage: the lytic killing of the mutant Escherichia coli W-518! Further, amongst the cellular debris, in the plaques where bacteria had been lysed to death, elements of the resistant W-518 bacteria emerged, which had survived the bacteriolytic onslaught.

When Lederberg examined these variant W-518 bacteria, the phage-infection survivors, she found that the bacteria became harbingers of the λ phage in the bacterial configuration called a lysogen. During the mixing experiment between the W-518 mutant (sensitive to death by phage λ) and parental K-12 (lysogenic and resistant to death by phage lysis), the W-518 mutant was also converted into a lysogenic state. In this newly acquired lysogenic state, the W-518 strain became a living host of the phage λ, like the original parent, Escherichia coli K-12, had, but not until W-518 and K-12 cells had been combined first in a test tube.

When Lederberg examined the interior of the lysis plaques that had emerged on the agar plates during the W-518 and K-12 bacterial combination experiment, she found the phage λ was present in the cell lysis debris. The Escherichia coli K-12 strain had long been thought to be a lysogen, but no one had tested this possibility until Lederberg had conducted the famous “accident experiment.” Thus, Lederberg discovered the λ phage existed as a so-called prophage stage of the virus after mixing the lysogen (K-12) with the indicator W-518 mutant. The Escherichia coli K-12 lysogen harbored the prophage form of the λ phage. Thinking at first that in the lysogenic bacterium, the λ prophage was in the form of a plasmid DNA molecule rather than a virus, she and others were surprised to find that the prophage λ DNA was integrated into the genome of the lysogen carrier, i.e., Escherichia coli strain K-12, now a famous bacterium.

Lederberg found that if the lysogen were exposed to minute amounts of UV light, the phage carrier, the lysogen, could be induced to regenerate the infectious form of the phage, converting the prophage back into a bacterial killer by lysis. In time, because Lederberg discovered bacteriophage λ, the virion would be used to discover the role of various genetic switches to regulate gene expression. The study of λ phage would permit investigators to understand the transduction process and permit gene cloning and DNA recombination.

2) Working backward—where and when was Lederberg born, and what do we know about her parents?

Esther Miriam Zimmer Lederberg was born on December 18, 1922, in the Bronx, New York. She and Benjamin, her younger brother, grew up during the Great Depression, and it is reported that her lunch sometimes consisted of bread drizzled with the juice of a freshly squeezed tomato.

Lederberg’s parents were David Zimmer, a Romanian immigrant who managed a print shop, and Pauline Geller Zimmer. She had a loving bond with her grandfather, who taught her Hebrew to perform the readings during the Passover Seders.

Lederberg graduated from high school at 16 and then enrolled at Hunter College, City College of New York, with a scholarship. She worked as a research assistant with Alexander Hollaender. That laboratory later became Cold Spring Harbor Laboratory in New York. Lederberg published her first paper on the genetics of the red bread mold, Neurospora.

After studying French and literature, she changed her major to biochemistry and graduated cum laude at the age of 20 in 1942. At first, her advisors were not supportive of her change in career paths because of the limited options available to women scientists at that time. Careers in science were primarily men-only occupations. From there, she won a fellowship at Stanford University, where she planned to study genetics. Lederberg supplemented her income by being a laboratory teaching assistant for George Beadle, washing her property owner’s clothes, and tidying up around the house. She recalls resorting to eating frog’s legs remaining from laboratory dissections due to not having money to buy food.

By 1946, Lederberg completed her Master’s degree at the age of 24 and then spent the summer at Hopkins Marine Station studying microbiology. Afterward, she moved to the University of Wisconsin to join her husband, Joshua, whom she married that same year. Lederberg began her doctorate with the assistance of a United States Public Health Service Fellowship and completed her Ph.D. degree requirements in 1950. The focus of her research was the bacterial species Escherichia coli.

Lederberg’s death was reported on November 11, 2006. She died in Stanford, California, at her home, due to pneumonia and congestive heart failure at the age of 83. Lederberg’s brother Benjamin and her second husband, Matthew Simon, survive her.

3) Fertility Factor or F Factor—again—why important, and how did this impact the world?

In later work, Lederberg mapped the insertion of the λ prophage genome into the lysogenic bacterium’s genomic site, near the gal locus, a genetic element encoding the metabolism of the sugar galactose. Integration of the λ phage’s genome into the bacterial host’s genome provided an opportunity for the bacteriophage to send its genetic instructions into new generations of Escherichia coli without having to kill the bacteria to do so. Lederberg would discover another famous method used by the λ phage to help the genetic transfer of phage from bacterium to bacterium and from generation to generation: the Fertility Factor F.

Bacteria that harbor these F factors can transfer genetic material that encodes the conjugation pili, and, importantly, the F+ bacteria take along new genetic elements as they make their way into new F˗ recipients. For example, genes that confer metabolism of sugars, like lactose or galactose, could come along with the F factor DNA from the donors to the F˗ females, conferring new metabolic features, which could be used to identify the new conjugants. Another new development involved using the F factors to recombine and transfer antimicrobial resistance genes between bacteria, which were also used as genetic markers and could be used for gene cloning.

The F factors were also used to study the genetics of new mutants. For example, investigators could map the locations of genes along the bacterial genome by inserting the F factor DNA into various chromosomal locations, interrupting specific genes, losing the genes’ encoded functions, and determining the nature of the lost functions. Then, by mating the mutants with bacteria harboring the wild-type genes on the F factor, the lost functions could be rescued, and the genetic determinants could be studied biochemically as well as genetically. During genetic recombination, Lederberg found that when the F factors left the bacterial genome, a segment of the bacterium’s DNA would be carried along with the exiting F factor DNA, producing a variant called the F’ factor (called F prime factor). Likewise, occasionally an F factor could leave behind a portion of its F factor DNA in the bacterial genome. With the advent of restriction enzymes, the various genes along the F factor DNA could be cut and spliced into new locations for mapping, gene cloning, or nucleotide sequence determinations. Thus, Lederberg’s pioneering studies involving the F factors provided a novel means for advancing the progress of molecular biology.

4) Her foundational studies—where did Lederberg study, and where did she get her Ph.D.?

As we mentioned above, Lederberg was the first investigator to isolate the famous λ bacteriophage as a graduate student. In her bacteriological-based experiments, she provoked the lysogenic bacterium Escherichia coli strain K-12 to produce a hitherto latent form of the λ phage from these cells. In so doing, Lederberg became the first scientist in history to discover the critically relevant viral microbe, the phage λ.

Lederberg’s famous discovery of the F factor came about using a cell mixing method similar to the combining experiment, where she mixed a phage-sensitive cell with a lysogenic strain, a method she used when she discovered bacteriophage λ. Lederberg had become interested in studying mating compatibilities, i.e., fertility, between various strains of bacteria. Those incompatible strains were deemed F˗ as a sexual cross between them were considered infertile or sterile. However, when Lederberg crossed a sterile bacterium with a fertile one, the infertile bacterium occasionally became F+, too, and could readily harbor the F factor. Lederberg published these findings in several papers in the early 1950s.

During the process of conjugation, a mechanism of DNA transfer between cells called bacterial sex or mating, the F factor, as the genetic element was known, plays an important role. The F factor DNA encoded a conjugation pilus that enabled the donor bacterium (called male or F+) to mediate transfer of its DNA in the form of a plasmid into a recipient bacterial cell (called female or F˗), converting the recipient into another F+ bacterium which could start the conjugation process over. At first, these transferrable genetic materials were called episomes and later called plasmids.

Lederberg was a prominent investigator in the invention of the replica plating technique. See Figure 1, in which clones that are resistant or sensitive to an antibiotic can be copied onto new plates, as shown. In their publication, the wife and husband Lederberg team reported their famous experiment as follows. First, they spread Escherichia coli cells onto a series of so-called master plates, and they incubated the cultures for several hours in a 37 °C incubator, permitting the plated bacteria to form small colonies. Next, they used a new device that they constructed: a square-cut velvet (velveteen) cloth wrapped around cylindrical wood or cork made sterile by autoclaving, Figure 1. With their sterile velveteen-cylinder apparatus, they merely touched the velvet to the master plate, keeping track of the plate orientation by marking the plates in one central location or using pins that dug into the new agar plates. The velveteen cylinder invention picked up the growing bacteria from their master plate. Then, Lederberg carefully used the inoculated velveteen cylinder to carry over to new plates containing phages, touching the newly inoculated plates, and applying the bacteria to the phage-laden plates and incubating them overnight at 37 °C.

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Figure 1. Diagram of the replica plate process.

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Escherichia coli cells that grew on the replicated plates were deemed resistant to lysis by the phage and could be picked from the master plate (or the replicate plates) to culture for study in pure form. Likewise, Lederberg could examine bacteria susceptible to phage infection by picking from their master plate the original bacterial colonies that had been lysed on the replicate plate.

The Lederbergs published their groundbreaking invention, the replica plate method, in the Journal of Bacteriology in March of 1952. The method in modern times does not differ by much and is considered a standard method for examining a variety of bacterial mutants. The Lederberg replica plating technique is regularly included in textbooks covering modern microbiology and microbial physiology, and biochemistry.

5) One caveat—Lederberg worked with her husband Joshua and never received the recognition that she deserved. Your thoughts?

At the age of 23 years, Esther Zimmer married Joshua Lederberg several months after taking her M.A. degree from Stanford University in 1946. According to Joshua, they honeymooned at a scientific conference, the American Association for the Advancement of Science, in late 1946 in Boston, MA. Esther Lederberg’s graduate studies concerned the effects of mutations in the fungus called Neurospora on para-aminobenzoic acid physiology. Lederberg had moved to the Osborn Botanical Laboratory at Yale University to be with Joshua. At Yale, Lederberg worked in the laboratory of Norman Giles, continuing with her interest in Neurospora genetics. While at Yale, Lederberg turned down a tuition grant from Columbia University to be with Joshua, who had taken a professorship at the University of Wisconsin at Madison. As a doctoral student, Lederberg would study Escherichia coli and genetic control mutants.

While enrolled in the doctoral graduate program at Madison, Lederberg’s research studies led to the discovery of bacteriophage λ, novel studies on the nature of phage lysogeny, and her groundbreaking work with the famous conjugation F factor in bacteria. In 1950, she had published separately on her discovery of bacteriophage λ. The Lederbergs had commenced collaboration, publishing their first paper together in 1951, detailing their studies on genetic recombination of Escherichia coli as a mechanism of heredity. They had mapped the genes for resistance to streptomycin and fermentation of the sugars called xylose and maltose. This work by the Lederbergs constituted a significant contribution to genetic recombination molecular biology.

In 1952, the Lederbergs published their famous paper using Esther’s replica plating invention to address the nature of bacterial mutations, supporting the contention by Luria and Delbrück that spontaneous mutation and natural selection played significant roles in evolution. They also reported on their collaboration with high-frequency recombination (Hfr) strains of Escherichia coli in which their F factors failed to transfer to F˗ recipients by conjugation. They later speculated that a yet undiscovered bacterial sex controlling factor, called an infective factor, was responsible.

In January 1953, they published a more extensively detailed paper regarding lysogeny but referred to Esther’s λ bacteriophage discovery only as an aside and without citing her earlier publications on her pioneering breakthroughs on phage λ and replica plating. The 1953 paper did cover the mixed culture analyses carried out by Esther, personally having tested about 2,000 clones of Escherichia coli mutants. Interestingly, without mentioning DNA specifically, they stated that a chromosomal factor was altered during the lysogeny of a bacterium by bacteriophage—the double-helix model of the DNA structure would not be published until later that year.

According to one source, a chapter on Lederberg in Women in Microbiology, published by the ASM Press in 2018, she was not appropriately credited for her work involving replica plating and other significant discoveries in which she was involved. One of these encompasses her publication of the preadaptation work, described in detail below. Another project that she was reported to have been involved in, but received neither authorship nor credit, was the discovery of the temperate bacteriophage called P22, a virus that participates in generalized transduction. This process transfers any gene between Escherichia coli donors and recipients. See Figure 2.

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Figure 2. Schematic illustration of an Enterobacteriae-specific phage virion called P22 (cross-section and side view).

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In 1956 Esther Lederberg and her husband participated in a project dealing with bacterial transduction and genetic recombination by phage λ, mapping the insertion of prophage DNA into a region near the gal gene of the Escherichia coli genome by analyzing thousands of individual recombinants. The Lederbergs published several papers on the mechanism of λ transduction in Escherichia coli. Throughout the 1950s and mid-1960s, the Lederbergs worked together. She performed much of the experimental laboratory work, giving Joshua time to write and publish numerous papers, but as a sole author.

6) Plasmids—what are they, and why did she study them and devote so much time to them?

Plasmids are short stretches of circularized DNA molecules. See Figure 3. These extra-chromosomal elements of DNA have been found occurring naturally as residents within cells of bacterial microbes, replicating on their own and independently of the genomic DNA synthesis. Many naturally occurring plasmids encode one or a few genes, each of which can supply an added property beneficial to the bacterium harboring the plasmid. Frequently, plasmids can confer a specific evolutionary advantage that permits survival, a selective enhancement in a new environment, or the propagation of genetic elements into the next generation of organisms.

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Figure 3. A bacterium is shown harboring genomic DNA and plasmids within the cytoplasm.

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For example, consider a scenario where a bacterium has encountered an excess source of a specific sugar in its environment and happens to have a plasmid that contains one or more sugar fermenting genes. The plasmid-harboring microorganism can express the machinery from the plasmid by making mRNA and protein to take advantage of the excess sugar. Keeping such a valuable plasmid would be in the bacterium’s best interest as it provides a distinct advantage in permitting its survival in such an environment. Thus, DNA plasmids can provide a specific degree of evolutionary adaptation, permitting an organism to acquire a distinct advantage and grow well beyond that degree conferred by the normal genes on its genome. This selective advantage afforded by the plasmid DNA on the microbe often ensures that the extra-chromosomal element is maintained within the microbial cell. The functional advantage, e.g., metabolism of sugar for energy, provided by the plasmid ensures its survival, in addition to the survival of the microbe that harbors the plasmid.

Plasmids that are artificially produced in the laboratory can also be employed to produce needed biological functions and products that may be needed in medicine or biotechnology. For example, specific plasmids, called cloning vectors, harbor reporter genes that permit the investigator to know which bacterial cells have them. These plasmid-based cloning vectors also contain so-called multiple cloning sites, consisting of a set of restriction enzyme-based DNA cutting sites that investigators can use to insert novel genes of interest for gene cloning purposes.

Further, specific plasmids permit the synthesis of sequencing templates for DNA nucleotide sequence determinations. These sequencing vectors were used quite effectively to complete the human genome project and the projects dealing with various genomes of many organisms, including viruses, bacteria, fungi, algae, plants, animals, and even individual human beings.

Other plasmid vectors have genetic elements that permit the expression of novel genes, permitting the transcription and translation of the genes of interest for manufacturing the desired gene product. When investigators studied the biology of the plasmids, biotechnology emerged as a predominant field in molecular biological circles. After a specific gene that confers a desired protein is cloned, sequenced, and expressed, the plasmids could be used to mutagenize the gene to manufacture gene products with better biochemical or therapeutic activities. What is more, mutagenesis can be readily utilized to alter the specificity of the gene product such that newer desired substrates can be metabolized more efficiently. Alteration of the substrate specificity in a protein can significantly benefit the fields of genetics, biomedicine, biotechnology, biochemistry, molecular biology, cell biology, immunology, vaccine biology, virology, bioinformatics, and medicine.

Lederberg’s studies of the F factor in Escherichia coli bacteria were groundbreaking in molecular genetics. She showed that insertion of the F factor was targeted to a specific genomic locus on the chromosome of the bacterium. She also learned that the genetic material became an independent plasmid-like molecule when the F factor DNA left the bacterium’s chromosome. The F factor behaved as an episome, i.e., a DNA molecule that could alternately exist as an integrated element within the bacterium’s genome or as a separate plasmid molecule replicating independently. See Figure 4.

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Figure 4. This diagram shows non-integrating plasmids (top) and episomes (bottom) within bacteria.

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Lederberg had been interested in plasmid biology, curious about the nature of the extant plasmids and their molecular properties. In 1974, she conducted a groundbreaking experiment in which she had transformed the cells of Escherichia coli and the bacterium Salmonella enterica serotype Typhimurium with a plasmid that transformed in a highly efficient manner. The plasmid, called pSC101, named after Lederberg’s colleague Stanley N. Cohen, encoded resistance to the antibiotic tetracycline. When Lederberg and Cohen modified the steps and amounts of magnesium and calcium salts in the solutions, the bacteria became highly competent in their uptake of the plasmid DNA molecules, and they observed a significantly higher frequency of resulting transformants. They published the work in the Journal of Bacteriology.

7) Some of Lederberg’s work built on Luria and Delbruck’s work—how do these ideas all fit together?

Lederberg used her replica plating invention to settle controversy regarding the famous fluctuation experiment conducted by Salvador Luria and Max Delbrück on the nature of the driving force for mutations in microbes. In 1942, Luria and Delbrück had speculated whether a specific environmental condition induced a mutation in an organism, which enabled it to grow better, or if the environment’s condition selected amongst a population of variants a spontaneous mutant that enabled the improved growth. The two hypotheses, i.e., mutations, were induced versus naturally selected among a population of spontaneous mutants, were hotly debated.

The mutation induction idea that the elements in the microbe’s environment caused the mutations predicted that culturing the microorganisms, such as bacteria, in the presence of the substance would generate equal numbers of resistant mutants in many cultures of the microbes. Likewise, without the mutation-inducing substance, there would be no mutants generated.

On the other hand, if the idea were accurate that selection occurred of pre-existing variants in a population of microbes, it predicted that a fluctuation in the number of resistant mutants would arise in a large number of cultures. Furthermore, the spontaneous mutant idea predicted that fluctuation in resistant mutants would occur with or without the environmental substance.

Luria and Delbrück observed a tremendous fluctuation in phage-resistant mutant numbers among the cultures they analyzed, with or without the phage. Luria and Delbrück published their pioneering results in the journal Genetics in 1943. However, their work was ignored, not understood due to the mathematical treatment given to their fluctuation data, or not believed by many scientists of the day. Most so-called non-believers had still held to the inaccurate notion of Lamarckian genetics—the idea that parental organisms acquire inheritable phenotypic traits if an inducing substance is present in that environment—that is, they still supported the false directed mutation hypothesis.

In 1952, the Lederberg’s would settle the matter by using her replica plating invention to study the evolutionary origins of bacterial mutations. The Lederbergs considered one of these phenomena “preadaptation,” the notion of spontaneous mutation and natural selection. Conversely, they called the opposing view, the idea that exposure to an environmental substance-induced mutation, as “directed mutation.” They reasoned that resistance of the Escherichia coli bacterium to T-1 phage infection would follow a mutant fluctuation pattern like the observations of Luria and Delbrück as predicted by the two opposing hypotheses.

That is to say, the Lederberg’s deducedthat Escherichia coli resistance to T-1 phage infection would arise spontaneously and randomly without needing the phage to be present to do so if Luria and Delbrück were correct about preadaptation. Their data predicted a so-called clonal occurrence of resistant mutants rather than a random sampling distribution. The Lederbergs deduced that they could test the preadaptation versus the directed mutation theories using her replica plating invention. Further, they figured astutely that the replica plating method permitted a direct demonstration of the clonal occurrence of antibiotic-resistant bacterial mutants, as predicted by Luria and Delbrück.

Thus, the experiments of the Lederbergs were conducted in two approaches. First, they examined phage resistance in mutants of Escherichia coli. Second, they examined the resistance of the bacteria to the antibiotic streptomycin. Let us consider the phage resistance phenomenon first.

In the Lederberg laboratory, Escherichia coli was cultured to saturation, diluted in broth, plated onto agar Petri dishes, and incubated, producing colonies on the plates. Then, as described above in Figure 1, Lederberg used her replica plating technique to make the replicates, i.e., “copies” of these plates of bacteria onto brand new plates. These new plates, however, contained about a billion T-1 phage viruses. After incubating the mixtures of Escherichia coli and T-1 phages, the plates would have developed a lawn of the bacteria. However, because of the high concentration of phages, the lawn was essentially reduced to battlefield surface of lysed Escherichia coli—a veritable landscape of bacterial debris.

In contrast, in places where T-1-phage-resistant Escherichia coli mutants were present, these variants grew to form well-developed colonies, entirely resistant to the deadly effect of the phage T-1. The replica plating technique allowed Lederberg to readily isolate these resistant mutants from the plates and grow separately in culture to purify them without exposing the bacteria to the phage as a selective agent. As predicted by the preadaptation hypothesis, the clonal characteristic of the phage-resistant mutants was thus directly supported by the data generated from Esther Lederberg’s replica plating method.

In the second set of experiments to test the preadaptation versus the directed mutation hypotheses, the frequency of streptomycin resistance in Escherichia coli was examined next by the Lederbergs. The strain of Escherichia coli K-12 used, called W-1, was wholly susceptible to the deadly effects of the streptomycin antibiotic, but the mutation rate was exceedingly low. Thus, to enhance the production of the streptomycin-resistant Escherichia coli K-12 W-1 mutants, Lederberg cultured the Escherichia coli K-12 W-1 strain in a large volume of broth. Then, after the new growth, an inoculum was transferred to a large volume culture flask. This process of successive passaging of the Escherichia coli W-1 was repeated several times. Then, after the inoculants were concentrated into a large batch, the concentrated Escherichia coli W-1 cultures, presumably containing mutant variants, were plated onto eosin-methylene blue (EMB) agar plates containing a high concentration of streptomycin (200 μg/mL). The streptomycin antibiotic concentration was high enough to kill any susceptible Escherichia coli K-12 strain W-1 cells but retain any streptomycin-resistant mutants.

After billions of Escherichia coli K-12 wild-type cells were plated, only about three resistant mutants were observed on roughly two dozen plates. The bacterial streptomycin-resistance mutation rate was meager. When the Lederbergs examined over 100 generations of Escherichia coli, they succeeded in isolating only about 240 streptomycin-resistant mutants. Unfortunately, this low mutation rate was insufficient to examine the clonal occurrence phenomenon predicted by the preadaptation hypothesis.

Thus, from a colleague, Henry P. Treffers from Yale, the Lederbergs borrowed a new Escherichia coli K-12 clone, strain 58-278, which was known to mutate faster than their W-1 strain. Next, after culturing the Escherichia coli K-12 strain 58-278 in large broth batches as before, without streptomycin, the Lederbergs plated the cultured strain onto plain agar and incubated the plates to permit any colonies, whether mutant, of Escherichia coli K-12 to form. The EMB agar was used on their master plates, where all related and unrelated enteric cells could grow, whether streptomycin-susceptible (parental and variant) or streptomycin-resistant (variant). Then, using their legendary replica plating method, Lederberg produced copies of each plate, transferring colonies from their master plate onto new Petri dishes containing about 200 μg/mL streptomycin in EMB agar. Any spontaneously occurring resistant variants (preadapted) would grow on the replica plates.

On the other hand, Escherichia coli K-12 cells that were streptomycin-susceptible did not grow on these plates. Whether Escherichia coli K-12 grew, i.e., regardless of their susceptibility or resistance to streptomycin, the original cells could be taken from the master plates and studied further if needed. The Lederbergs observed clearly that the frequencies of streptomycin-resistance occurred entirely independent of any exposure to the selective agent—streptomycin—thus, it was clear that the streptomycin-resistant bacteria had arisen spontaneously. Their growth on the EMB plates with streptomycin showed that they were already present in a population of cultured Escherichia coli K-12 cells. The data, thus, supported the preadaptation hypothesis. The Lederbergs, Esther and Joshua, published the work together in the prestigious Journal of Bacteriology in March of 1952.

8) Lederberg also initiated the specific system of naming what we call insertion sequences. Why is this important?

In the mid-1970s, Lederberg became the director of the Plasmid Reference Center in California at Stanford University’s School of Medicine. The new institution was devoted to archiving plasmids, both natural and artificial, in an organized repository. The archival effort established the naming nomenclature of the archived plasmid molecules. Furthermore, the Plasmid Research Center would distribute desired plasmids to investigators who requested specific recombinant plasmids for research at other institutions.

Soon after assuming the directorship, Lederberg expanded the plasmid archival program to include transposons, known as “jumping genes” discovered by Barbara McClintock, and mobile genetic elements harboring so-called insertion sequences. The transposons and insertion elements move from one genetic location to another, sometimes within the same DNA segment or on a different segment.

The insertion elements are simple transposable DNA molecules flanked by so-called inverted repeat nucleotide sequences that transposase enzymes target. Alternatively, transposons can harbor and carry with them additional genes, like those for fermentation or antibiotic resistance, conferring these new properties upon the recipient transposants of the new movable genetic elements. As the Center acquired the transposons and insertion sequences, Lederberg began numbering them sequentially, using the designations IS and Tn, followed by their specific numbers. For instance, the insertion sequence called IS1 consists of the transposase enzyme gene and two flanking inverted repeats. See Figure 5. On the other hand, the transposon Tn10, for example, harbors the flanking IS elements and contains a gene that confers resistance to tetracycline.

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Figure 5. Composite transposon.

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9) After several years, she divorced Joshua, her husband, who had been claiming much of the credit for her work—has she ever really been acknowledged for all she did?

In 1958 Joshua Lederberg, Edward Tatum, and George Beadle were awarded the Nobel Prize in Physiology or Medicine. While Tatum and Joshua acknowledged Esther Lederberg early in the press briefings and Nobel festivities, at the ceremony itself, she had been relegated to the role of the “laureate’s wife” rather than as an expert bacterial geneticist who had been involved in many of the laboratory experiments dealing with phage λ lysogeny, genetic recombination, F factor conjugation, and phage transduction.

Shortly after the Nobel, Lederberg followed Joshua to Stanford University, where he had been appointed chair of the Genetics Department. She took an untenured position in the Medical Microbiology department as a “research professor.” This offer was made only after extensive prodding of the dean.

Altogether, the Lederbergs published about a dozen papers together. Their last collaboration had dealt with the nature of suppression by streptomycin of mutations in the gal operon of Escherichia coli, publishing the results in their last paper together in 1964. The Lederbergs were to divorce two years later. While staying at Stanford, her salary originated solely from external grants. Esther Lederberg never received tenure at Stanford.

One of her collaborators, Luigi Cavalli-Sforza, wrote in 1974 of Lederberg that she had never been given proper credit for which she had been so deserving because “her husband had been very famous.” Unfortunately, in later years, in 1987, Joshua wrote an autobiographical account in the prestigious Annual Review of Genetics on the genetic recombination discovery without mentioning Esther by name or as a direct contributor.

10) What have I neglected to ask?

It was difficult for Esther Lederberg to be a master’s graduate student in the mid-1940s with little or no funding. She arrived at Stanford, CA, with no luggage as it was lost en route and unwittingly stayed in a motel of a dubious nature in Palo Alto. With an interest in genetics, she entered Edward Tatum’s laboratory.

Stanford’s climate was considered a sink or swim type of scenario, especially in genetics research. With funding prospects unavailable for Lederberg, she had to take on odd jobs to pay the rent, such as doing an entire family’s laundry in exchange for her lodging in the house. At Stanford, Lederberg and a fellow graduate student resorted to eating the left-over frog legs after completing the laboratory courses!

Early on in her scientific career, in 1952, Lederberg studied elements of the not-yet-famous lac operon. She studied recombinant mutants that became Lac+ as they could metabolize the milk sugar lactose for energy and growth after she crossed two mutants that were Lac˗. To obtain a Lac+ recombinant, Lederberg had tested over 30,000 combinations! She had published the work in the journal Genetics concluding that her revertant mutants, i.e., those that went back to Lac+, showed a stable form as well as an unstable suppressor form that could readily circumvent a defective Lac˗ mutation. As the rest of Lederberg’s career would show, she was always ahead of her time.

Esther Lederberg was a performing musician, playing concerts devoted to ancient music, such as Baroque-era classical music, medieval music, and Renaissance period music, each with historically accurate instruments. She had helped to establish a group in 1962 called the Mid-Peninsula Recorder Orchestra. Lederberg’s musical proclivity led to her encounter with a musical aficionado, an engineer named Matthew Simon. With their shared interest in ancient music as a starting point, they were married in 1989 and stayed together until her death from congestive heart failure, a complication of pneumonia, on November 11 in 2006, at 83 years old.

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