Module 1.1.1

Escherichia coli and the French School of Molecular Biology

AGNES ULLMANN

Posted January 25,2010

Institut Pasteur, 75015 Paris, France

Mailing address: Institut Pasteur, 28 rue du Dr. Roux, 75015 Paris, France. Phone: 33 1 45 68 83 85, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

THE ROOTS

The rise of biochemical genetics started with the “one gene-one enzyme” theory, which stated that one gene controls the production of one enzyme that affects a single step in a metabolic pathway (3). However, the development of what we now call molecular biology originated in the creation of the “Phage Group” in 1940 by Max Delbrück, Salvador E. Luria, and Alfred D. Hershey at Cold Spring Harbor, which greatly influenced the future French school of molecular biology (39). Jacques Monod, one of the founders of that group, recalls in his Nobel Lecture (34) the great revelation he experienced by the end of World War II, when, in an itinerant library of the American army, he discovered the Luria-Delbrück paper (22) on the spontaneous character of some bacterial mutations. For Monod this paper represented the birth of bacterial genetics. While he still was in the French army, Monod also discovered the epoch-making publication of Avery, MacLeod, and McCarthy that identified the transforming principle as deoxyribonucleic acid—another fundamental revelation (2).

While Europe was torn by war, in the United States, Delbrück, a German quantum physicist, and Luria, an Italian medical doctor, decided to collaborate on the nature of phage resistance in bacteria. They wanted to answer the question whether resistance resulted from mutation followed by selection or from adaptation induced by exposure to the phage. The idea of the experiment, later called “fluctuation test,” came to Luria at a faculty party, when he watched the fluctuating returns obtained by colleagues playing on a slot machine (19). The next day, he conducted the experiment: he inoculated a small number of Escherichia coli into separate culture tubes. After a certain period of growth, equal volumes of these separate cultures were added to plates saturated with T1 phage. If phage resistance began only at the moment of exposure of the bacteria to the phage, then all plates should show the same distribution of resistant clones. The experiment showed a large fluctuation from the average count, providing evidence that phage-resistant mutants originated by spontaneous mutation and not as a result of phage attack. If mutation occurred early after inoculation, then a large number of resistant cells (called “jackpot”) would be present in the population, whereas smaller numbers would be present if the mutation occurred later during growth. That was precisely the outcome of the experiment, and the paper by Luria and Delbrück was published in 1943 (22). In 1969 Luria and Delbrück, together with Hershey, were awarded the Nobel Prize for Physiology or Medicine for work on the replication mechanisms and genetics of viruses.

THE BEGINNINGS OF THE FRENCH SCHOOL

The three founders of the French school of molecular biology were André Lwoff (1902–1994), Jacques Monod (1910–1976), and François Jacob 1920– ) (Fig. 1, Fig. 2, and Fig. 3).

Fig. 1André Lwoff.

Fig. 2Jacques Monod.

Fig. 3François Jacob.

Lwoff started his scientific career in 1922 at the age of 20, when he entered the Pasteur Institute in the laboratory of Felix Mesnil (a secretary of Louis Pasteur), where he devoted his work to the nutrition of ciliates. This seemed to be an enormous task at that time, because no pure cultures in synthetic media had been previously obtained. After he finally succeeded, he went on to study the nutritional requirements of some protists, and realized that growth factor requirements could be interpreted as a loss of some biosynthetic functions, that is, as an evolutionary consequence of adaptation to parasitism (23). His theory of regressive physiological evolution conflicted with the dominant ideology of that time, implying that progressive evolution is linked to enrichment of functions. In his introduction to “L’évolution physiologique,” Lwoff relates a discussion that he had with one of his colleagues who accused him of being ignorant about evolution, and arguing that he (the colleague) was able to do things that a Chlamydomonas would be unable to do. Lwoff concluded by wondering how this eminent colleague would have survived, exposed to sunlight in a potassium nitrate solution (24)?

One of Lwoff's major discoveries during this period was the demonstration that a vitamin may function as a coenzyme, which led to the recognition of their role in cellular metabolism, and to the conclusion that vitamins constitute a class of essential growth factors for organisms that are unable to synthesize them (27).

During World War II, Lwoff's laboratory (the “Service de Physiologie Microbienne”), the famous attic, became an active center of the underground. Jacques Monod, before definitively joining his laboratory in 1945, spent some time there clandestinely to perform a few experiments on “enzymatic adaptation.”

Monod started his scientific career in 1931, working on ciliates, organisms that soon did not satisfy his curiosity. In the summer of 1934 he embarked on Captain Charcot's ship the Pourquoi pas? (Why not?) for an ethnographic mission in Greenland and studied the natural history of the region (Fig. 4). In the spring of 1936, Monod was preparing to take part, for the second time, in an expedition to Greenland, but Boris Ephrussi, a French geneticist of Russian origin, who was going to spend a year with T. H. Morgan's group, diverted him from this project. Ephrussi convinced Monod that genetics was important and helped him to obtain a Rockefeller Fellowship; they both went to Pasadena (Fig. 5). At the same time, the Pourquoi pas? was caught in a violent storm on the coast of Greenland and was lost with all the crew. Genetics had saved the life of Jacques Monod, a debt of gratitude that he would later repay.

Fig. 4Jacques Monod on Pourquoi pas? (Greenland, 1934).

Fig. 5Boris Ephrussi and Jacques Monod on the roof of Caltech (1936).

Monod started to work on bacterial growth in 1937, following the advice of Lwoff. At that time, he was already assistant professor in the Zoology Laboratory at the Sorbonne where he began to grow E. coli in different synthetic media. By using quantitative analysis of how populations of single cells grew, he was able to show that the growth rate was a function of substrate concentration. In 1940, he made his first remarkable discovery, i.e., the phenomenon of diauxy (32). After having grown bacteria in the presence of one carbohydrate, Monod found it interesting to study the interaction of two carbon sources. In some mixtures of two sugars, when one of them was glucose, he observed two distinct growth cycles, separated by a lag phase. This he called diauxy (33) (Fig. 6). Monod recalled how, in December 1940, he went to the Pasteur Institute to show Lwoff the diauxic curves and asked what could that mean? When Lwoff answered that it could have something to do with enzymatic adaptation, Monod asked him: “Enzymatic adaptation, what is that?” Lwoff's intuition was correct: the diauxic growth was the result of the inhibitory action of glucose on the formation of adaptive enzymes that metabolize the second carbohydrate (today, we call this catabolite repression [31]), and the lag phase corresponds to the time necessary for the induction of these enzymes. The concept of induced enzyme synthesis was born. According to Monod, this 1940 visit to Lwoff's laboratory was the turning point of his life, and from then on all his scientific activity was focused on the study of enzymatic adaptation (34). Nevertheless, further developments had to wait until the end of World War II. During the war, Monod took an active part in the underground, joined armed resistance movement in 1943, became chief of the National Headquarters, and after the liberation of Paris in 1944, he joined the free French forces where he had important responsibilities (Fig. 7).

Fig. 6Diauxic growth curves of E. coli (from reference 34). Growth of E. coli in the presence of different carbohydrate pairs serving as the only source of carbon in a synthetic medium.

Fig. 7Jacques Monod in the military (1944).

THE NATURE OF ENZYME INDUCTION

The war ended and Monod joined the “Service de Physiologie Microbienne” at the Pasteur Institute, where Lwoff had gathered a remarkable staff that included, besides Monod, Elie Wollman, Pierre Schaeffer, and later François Jacob. A great number of foreign scientists, including Melvin Cohn, Annamaria Torriani, Martin Pollock, Alvin Pappenheimer, Mike Doudoroff, Dale Kaiser, Dave Hogness, Germaine Cohen-Bazire, and many others joined Lwoff's laboratory. The “Golden Period” started.

Monod chose to study the adaptive enzyme, β-galactosidase, an enzyme that hydrolyzes lactose into glucose and galactose. Studying the increase of β-galactosidase activity after addition of lactose to a culture of E. coli (induction), Monod and collaborators (A. M. Pappenheimer, G. Cohen-Bazire, D. Hogness, and M. Cohn) were able to demonstrate that the increase in enzymatic activity during induction was a true measure of the total biosynthesis of β-galactosidase from amino acids (6, 9, 37). Once synthesized, the molecule of β-galactosidase was fully stable in vivo; this was the first breakthrough to the understanding of the nature of enzyme induction (36). In addition, these experiments dealt a severe blow to the Schoenheimer dogma of the “dynamic state of living matter.”

Until the beginning of 1950, only the substrates of enzymes were known to serve as enzyme inducers. One of the predominant hypotheses that had been proposed to account for the induction activity of the substrates was that the substrate-inducer combined with the precursor of the enzyme, thus shaping it in its active form. The way to test this hypothesis was to show that all substrates, as well as competitive inhibitors, were inducers. Next, Cohn and Monod decided to synthesize a number of lactose analogs; some of them (i.e., some thiogalactosides) turned out to be excellent inducers, without being hydrolyzed by the enzyme; they were called “gratuitous inducers.” Others were shown to be substrates without any induction activity (Fig. 8). It was around this period that Monod decided to drop the term “enzymatic adaptation” and use instead “induced enzyme synthesis,” a term that was adopted, as Mel Cohn recalls, “in an encyclical issued by the Adaptive Enzyme's College of Cardinals”: Cohn, Monod, Pollock, Spiegelman, and Stanier (7).

Fig. 8Comparison of various β-galactosides as substrates and as inducers of β-galactosidase. I, lactose: substrate of the enzyme, but deprived of inductive activity. II, methyl-β-d-galactoside: low-affinity substrate effective inducer. III, methyl-β-d-thiogalactoside: not hydrolyzable by the enzyme, but a powerful inducer. IV, phenyl-β-d-galactoside: excellent enzyme substrate, high affinity, no inductive ability. V, phenyl-β-d-thiogalactoside: no activity either as a substrate or as an inducer, but capable of acting as an antagonist of the inducer (from reference 34).

Monod realized that, for a better understanding of enzyme induction, he had to study the relationship between gene and enzyme. He isolated E. coli mutants unable to grow on lactose (Lac⁻), while some among these mutants were still able to synthesize β-galactosidase. These were the so-called “cryptic” mutants. The nature of these mysterious mutants was solved much later by Monod, Rickenberg, Cohen, and Buttin, who showed that labeled thiogalactosides accumulated rapidly, reaching up to 5% of the bacterial dry weight, in induced wild-type bacteria, but they did not accumulate in either uninduced ones or in the cryptic mutants (41). The conclusion was clear: the factor responsible for thiogalactoside accumulation could only be a specific protein, a pump, controlled by a gene, y, distinct from the β-galactosidase gene, z. The protein was named lactose permease; it became a novel category of proteins that mediate the penetration of small molecules into the bacterium. It took some time before the scientific community accepted the existence of the permease, based only on in vivo experiments; many scientists also objected that one should not give a name to a protein before it had been isolated. Nevertheless, it took 24 years for lac y to be cloned and sequenced (4), and another 23 years to obtain the X-ray structure of the lactose permease protein (1).

By further studying the relationships between gene and enzyme, in a few years, Monod uncovered the mechanism of β-galactosidase induction and established that in E. coli the synthesis of β-galactosidase depends on (i) on the gene, z, governing the capacity or incapacity to produce the enzyme; (ii) on the gene, y, governing the synthesis of lactose permease; and (iii) on a genetic factor, known to exist under the forms i⁺ (wild type, corresponding to inducibility) and i⁻ (corresponding to constitutivity). Genetic analysis revealed that the z, y, and i genes are closely linked on the E. coli chromosome (10).

In 1959, a new enzyme, galactoside transacetylase, was isolated by Zabin, Kepes, and Monod (44). It turned out that the corresponding gene (lacA) was genetically linked to lac y and lac z and subject to the same regulation.

LYSOGENY

While Jacques Monod was studying the specificity of β-galactosidase induction, André Lwoff started to work around 1949 on lysogeny. At the Pasteur Institute, the phenomenon of lysogeny has been pioneered by Eugène and Elizabeth Wollman; they worked on bacteriophages for 20 years before perishing in Nazi camps. When Lwoff took up the study of lysogeny, the first question he asked was how are bacteriophages liberated by lysogenic bacteria; are they continuously secreted or liberated at certain stages of the bacterial cycle? To answer this question, Lwoff chose a lysogenic Bacillus megaterium, a particularly large bacterium, because he wanted to study single bacteria. Eugène Wollman, with whom he had several discussions on lysogeny between 1930 and 1939, introduced him to this material. With a microscope and a micromanipulator he inoculated individual bacteria into individual microdrops and let them grow. Why this methodological choice? Because he “disliked mathematics, and wanted to avoid formulas, statistical analysis and, more generally, calculations as much as possible (26).” By following the kinetics of phage production, he realized that only a relatively small fraction of the bacteria lysed and produced bacteriophage. With Louis Siminovitch and Niels Kjeldgaard he tried, day after day, to obtain lysis of the totality of the bacterial population. After several unsuccessful months, he decided to irradiate bacteria with ultraviolet light—a UV lamp used by Jacques Monod to mutagenize E. coli, was available at the other end of the corridor. The bacteria were irradiated for a few seconds, and after one hour the culture was entirely lysed. He wrote, “As far as I can remember, this was the greatest thrill of my scientific career—for the first time in my life, I had the feeling of having discovered something (26).”

This was indeed a far-ranging discovery; a clear picture of lysogeny emerged. When a phage infects a bacterium it can enter either a lytic or a lysogenic cycle. The lysogenic bacterium perpetuates the genetic material of the bacteriophage (which Lwoff dubbed the prophage), while induction of the prophage (i.e., UV radiation) would lead to multiplication and release of the phage (25) (Fig. 9). In those years the concept of repression and derepression had not yet come to light. Nevertheless, as Lwoff recalls, Jacques Monod used to say that induction of enzyme synthesis and of phage development are the expression of one and the same phenomenon. This statement looked paradoxical but was, paradoxically, a remarkable intuition (26).

Fig. 9Lysogenic cycle (from reference 29).

BACTERIAL CONJUGATION

François Jacob joined Lwoff's laboratory in 1950. At the beginning of the war he was a medical student and wanted to become a surgeon. In 1940 he joined the free French forces in London, and after having participated in the Africa campaign with the division Leclerc, he was severely injured during the Normandy landing in 1944 (Fig. 10). Because of his injury he had to give up the idea of becoming a surgeon, but he had the chance to start research, as he recalls, at the good place just at the right time. He arrived just after the discovery by Lwoff of the induction of phage production. He started his research by a genetic analysis of lysogeny. With the discovery of bacterial conjugation by Lederberg and Tatum (21), as well as the discovery of mutants (Hfr) that transfer the genetic material to female bacteria (F⁻) with high frequency by Cavalli and Hayes (5, 8), the study of genetic determination of lysogeny took a completely different turn.

Fig. 10François Jacob in the military (1944).

Elie Wollman entered Lwoff's laboratory after World War II. After having spent two years in Delbrück's laboratory at Caltech working on bacteriophage T4, he returned to the Pasteur Institute in 1950. Probably, in remembrance to his parents, Eugène and Elisabeth Wollman, pioneers in studying lysogeny, he started to work on an E. coli K-12 strain, lysogenic for a phage called lambda, discovered by Esther and Joshua Lederberg (20). Wollman showed that the prophage could behave as a genetic marker and that it was linked to the gal locus on the chromosome (42).

Elie Wollman and François Jacob began a collaboration to study the genetics of lysogeny by using the E. coli K-12 lambda lysogenic strain. By doing crosses between lysogenic and nonlysogenic bacteria, they discovered a new phenomenon that they called “zygotic induction”: crosses between lysogenic Hfr and nonlysogenic F⁻ resulted in the induction of phage development, but not when the reciprocal cross was performed (15). This asymmetry in crosses suggested that the immunity of lysogenic bacteria to infection by the homologous phage is caused by a cytoplasmic factor preventing prophage induction; this factor was later called immunity repressor (16).

By analyzing the conjugation process itself between various Hfr and F⁻ strains, Wollman and Jacob used the technique of interrupted mating, in which a high-speed blender was used to separate mating pairs after different periods of time. They showed that the male chromosome was injected into the female at a constant rate; this enabled them to dissect the main events of bacterial conjugation and to construct a map of the bacterial chromosome by following the sequence of genes as a function of the time of entry (43). Further analysis of the conjugation process led Jacob and Wollman to demonstrate the circularity of the E. coli chromosome (18). By studying how the Hfr strain originates from F⁺, they showed that the integration of the F factor into the chromosome was a single mutational event. The F factor could, therefore, exist either as an autonomous element, controlling its own replication and transfer, or in the integrated state (Hfr), it was able to trigger conjugation and chromosome transfer. Jacob and Wollman called this class of genetic elements, which moved back and forth between chromosome and cytoplasm, “episomes” (17). The episome shared this property with the prophage that also could exist in an integrated or autonomous state. The term episome was replaced later by the term plasmid.

It was probably difficult to predict that the discovery of lysogeny by André Lwoff would change the subsequent development of the French school of molecular biology. The discovery of zygotic induction and the unraveling of the mechanism of sexual process in bacteria were direct consequences of this discovery. They provided new and powerful tools to attack the problem of regulation.

REGULATION

An extremely fruitful collaboration between Jacques Monod and François Jacob started in 1957 (Fig. 11). They decided to use a genetic approach, i.e., conjugation for a genetic analysis of the lactose system. The different i, z, and y mutants isolated by Monod were then inserted in various combinations in either male or female bacteria. With Arthur Pardee, who spent a sabbatical year at Pasteur, Jacob and Monod performed one of the most famous experiments in molecular biology, known as the PaJaMo (or PyJaMa) experiment (40). This experiment involved measuring the synthesis of β-galactosidase in zygotes resulting from the conjugation of male bacteria, carrying the z⁺ and i⁺ genes, with females, carrying the z⁻ and i⁻ genes. In the absence of inducer, none of the parents are able to synthesize the enzyme: the male, because of the absence of inducer, and the female, because of a defective z gene. Crossing the two strains, enzyme synthesis began within a few minutes after the z⁺ gene entered the recipient, but after an hour or so, enzyme synthesis stopped. When inducer was added, enzyme synthesis resumed, suggesting that the transferred i⁺ gene was becoming gradually expressed and the zygote became phenotypically inducible (Fig. 12). The PaJaMo experiment showed that the i⁺ gene is dominant over the i⁻ gene and led to the model of negative regulation: the i⁺ gene produces a factor that blocks the expression of the z⁺ gene. This factor was called “repressor.” The rapid expression of the z⁺ gene in the i⁻ cytoplasm was to become the experimental basis for the development of the messenger RNA model, which stated that the information from DNA was transferred first to a metabolically unstable RNA, that, in turn, would be translated into polypeptide chains on the ribosomes. This discovery gave a new impetus to the new field of molecular biology.

Fig. 11Jacques Monod and François Jacob (in approximately 1961).

Fig. 12The PaJaMo experiment (from reference 40) (redrawn by Jean Marc Ghigo).

In 1961, Monod and Jacob presented a model for the regulation of gene expression, the operon model (11) (Fig. 13). The concept of operon stemmed also from the concept of lysogeny; the analogy of the phenomenon of zygotic induction with the PaJaMo experiment was obvious. To account for the specificity of action of the repressor, a new structure was proposed: the “operator.” A single operator controlled the expression of the adjacent z and y genes of the lactose system in a coordinate manner. Mutations inactivating the operator—unable to recognize the repressor—were isolated. The operon, a unit of coordinate transcription, was born. An operon is composed of structural genes, connected to an operator, subject to the action of a repressor, produced by a regulator gene. The binding of an inducer to the repressor will disrupt its interaction with the operator, and transcription of a polygenic mRNA will take place (12, 13). A few years later, another indispensable element, the “promoter,” which was needed for expression, was identified as the site of transcription initiation (14).

Fig. 13The operon model. The lactose operon in the repressed (A) and induced state (B). In the absence of an inducer, the LacI repressor binds the operator and prevents lac gene expression, while in the presence of the inducer, the LacI repressor is inactivated and lac genes are expressed (drawing courtesy of Jean Marc Ghigo).

Within ten years, the problems posed by the induced synthesis of β-galactosidase in E. coli had been solved. The new ideas were applied to a large number of catabolic and anabolic pathways and to viral development. The nature of molecular communications was clarified.

ALLOSTERY

One of the major concerns of Monod was how proteins recognize chemical signals: how the repressor recognizes inducer, or how, in a biosynthetic pathway, the product of the last enzyme inhibits the activity of the first enzyme. At that time, Jean-Pierre Changeux, a student of Monod, studying threonine deaminase, showed that the substrate threonine and the inhibitor isoleucine seemed to be bound to different sites of the enzyme. Based on these and other findings, Monod developed one of the most elegant concepts of molecular biology, the theory of “allostery” (35). Allosteric proteins were postulated to possess at least two, stereospecifically different sites: the active site, which binds the substrate, and the allosteric site, which binds specifically to the allosteric effector (Fig. 14). Thus, the allosteric effects are entirely due to reversible conformational alteration induced in the protein when it binds to the specific effector. The concept of allostery was a major biological generalization and one of the most important ideas to emerge from the study of bacterial regulatory mechanisms (38). Allostery made possible the interpretation and integration of a great number of isolated observations into a coherent unifying concept. It was endowed with such explanatory power that it did not exclude anything. This is why Boris Magasanik called it “the most decadent theory in biology.” Jacques Monod was quite satisfied with this definition.

Fig. 14Model of allosteric transition produced in a symmetrical dimer. In one of the two conformations, the protein can attach itself to the substrate and to the activating bond. In the other conformation, it can attach itself to the inhibiting bond (from reference 34).

In 1965, André Lwoff, Jacques Monod, and François Jacob were awarded the Nobel Prize in Physiology or Medicine for discoveries concerning the genetic control of enzyme and virus synthesis (Fig. 15). One may ask what the impact of this epoch-making period was on modern science? The concepts developed during this period are absolutely central to modern biology. One of them, the regulation of gene expression—essentially the Jacob-Monod model—was a main forerunner of the biotechnological revolution, based on recombinant DNA techniques, and proved to constitute the basis of gene expression in eukaryotic systems. Messenger RNA, a concept derived from the operon model, became a tool in the modern microarray technology of hybridization, which uses nucleic acid probes for determining the presence of particular base sequences.

Fig. 15Lwoff, Monod, and Jacob in 1965.

β-Galactosidase, used by Monod as a paradigm of enzyme induction, has become a classical tool in modern biological research: lacZ used to be the most commonly used reporter gene in the analysis of developmentally regulated systems and tissue-specific expression. The concept of lactose permease, a membrane-associated protein that allows bacterial cells to pump β-galactosides from the medium, was the precursor of the widespread membrane-associated pumps that play important roles in biological phenomena.

We know today that most mechanisms of cell signaling involve allosteric interactions. The theory of allostery even inspired the prion theory, which implies a transmission of conformational change between identical protein molecules, as was postulated to occur between protomers in an allosteric protein.

The main importance of the French school of molecular biology probably was to profoundly alter the thinking of biologists. A number of factors played in this success. As André Lwoff pointed out: “The right problem was posed at the right time in the right environment. For an outsider, the success appears to be the highly improbable combination of improbable events. Yet one should not forget the numerous trials and errors and the fact that selection intervenes constantly at each step of the phylogeny of a scientific construction (28).”