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Escherichia coli and the Emergence of Molecular Biology

Agnes Ullmann

Posted 10 June 2011

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

 

PREHISTORY

In 1885, Theodor Escherich, a German pediatrician, isolated from the intestines of neonates bacteria that were gram negative and slightly motile and grew well on many of the artificial media available at the time. Escherich named these organisms Bacterium coli commune, known since 1919 as Escherichia coli.

In 1907, Rudolf Massini described an organism, named Bacterium coli mutabile, which he obtained as a strain that, while lactose negative on primary isolation, later developed papillae that did ferment lactose. This finding probably opened the way to many subsequent studies on E. coli leading to the emergence of molecular biology (30).

The roots of what we call today molecular biology can be found in the convergence of chemistry, biochemistry, genetics, and physics. Linus Pauling's contribution to our understanding of the chemical structure of proteins in the late 1930s was of great importance; during the same period, Beadle and Tatum's (Fig. 1) “one gene—one enzyme” theory revealed the importance of biochemical genetics (3). The theoretical physicist Erwin Schrödinger's book What Is Life?, published in 1944, is considered to be one of the most influential scientific writings of the 20th century. His suggestion that the heredity material behaves like an “aperiodic crystal” whose structure allows the encoding of a large amount of information was an inspiration for the future discovery of the structure of DNA. The book had an immense influence on the future leaders of the field of molecular biology; therefore, Schrödinger (Fig. 2) was considered one of the motivators of the birth of molecular biology.

A major contribution to the identification of the heredity material was the fundamental discovery made by Avery, MacLeod, and McCarthy in 1944. It was based on the experiment of Griffith, who showed in 1928 that an avirulent strain of pneumococcus (a “rough” bacterium) could become infectious when mixed with a virulent strain (with a “smooth” aspect) that had been heat killed. Griffith concluded that the dead bacteria provided some substance that transformed the harmless bacteria into the infectious ones. Avery and collaborators identified Griffith's transforming principle as DNA. They showed that DNA alone from smooth bacteria caused rough bacteria to become transformed and concluded that, indeed, DNA was the hereditary material (2).

 

THE BEGINNINGS

The development of molecular biology originated in the creation of the “Phage Group” in 1940 by Max Delbrück and Salvador E. Luria (Fig. 3), later joined by Alfred D. Hershey at Cold Spring Harbor. At the same time, Escherichia coli and its viruses became the materials of choice for basic studies in molecular genetics.

Delbrück, a German quantum physicist, arrived in the United States in 1937 as a Rockefeller fellow to study genetics. He went to the California Institute of Technology to work with Emory Ellis on bacteriophages. In 1939, they published a paper on phage growth, known as the one-step growth experiment, which provided a quantitative description of phage multiplication (14). Delbrück became one of the most influential persons within the wave of physical scientists who joined the field of biology during the 20th century. His contribution to the development of molecular biology was enormous.

Luria, an Italian medical doctor, left fascist Italy in 1938 for Paris. In 1940, he had to leave France and succeeded in going to the United States. Soon after his arrival he met Delbrück, and they decided to collaborate on the nature of phage resistance in bacteria. They wanted to answer the question as to whether resistance resulted from mutation followed by selection or from adaptation induced by exposure to the phage. The Luria-Delbrück experiment, known as the “fluctuation test,” consisted of the inoculation of a small number of bacteria (E. coli B) into separate culture tubes. After a certain period of growth, equal volumes of these separate cultures were plated onto dishes 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 roughly the same distribution of resistant clones. The experiment showed a large fluctuation from the average count, providing evidence that phage-resistant bacteria arose by random, spontaneous mutations of sensitive cells independent of the action of the virus. The experiment allowed, in addition, the determination of the mutation rate. The paper of Luria and Delbrück, published in 1943, certainly marks the beginning of modern bacterial genetics (28).

Alfred Hershey joined the “Phage Group” in 1943 after he met with Delbrück, who recommended him to Luria. This is how Delbrück described his first impression of Hershey in a letter to Luria: “Drinks whiskey but not tea. Simple and to the point. Likes living on a sailboat for three months; likes independence (24).” Hershey was already known for his work on crosses with the T-even phages of E. coli showing that genetic recombination takes place between phages present in the same cell. Yet his name is linked to the famous Hershey-Chase experiment carried out with Martha Chase in 1952 (Fig. 4). They labeled the DNA of T2 phages with radioactive phosphate, infected E. coli cells, and then removed the protein shells from the infected cells with a blender and separated the cells and viral coats. They found the presence of radioactivity only in the pellet of bacterial cells, not in the supernatant containing the protein shells. In a second experiment, they labeled the phages with radioactive sulfate. After separation, radioactivity was found in the protein shells but not in the infected bacteria, supporting the hypothesis that the genetic material that infects the bacteria is DNA (19).

Recollecting those early days, this is how Frank Stahl described the Phage Group: “The Phage Church, as we were sometimes called, was led by the Trinity of Delbrück, Luria, and Hershey. Delbrück's status as founder and his ex cathedra manner made him the pope, of course. Luria was the hard-working, socially sensitive priest-confessor, while Hershey was the saint (41).” The members of this Trinity were awarded the 1969 Nobel Prize for Physiology or Medicine for their discoveries concerning the replication mechanism and the genetic structure of viruses.

 

COLD SPRING HARBOR

Most concepts of the physical basis of heredity were born in the 1940s in Cold Spring Harbor, a small station of modest resources on the shores of Long Island Sound. Milislav Demerec (Fig. 5), the director of the Cold Spring Harbor Laboratory (CSHL) from 1941 to 1960, played a crucial role in its development. He recruited scientists to develop basic studies on the molecular genetics of bacteria and their viruses and was instrumental in providing housing and laboratory space for visiting scientists.

The station was also where the important “Cold Spring Harbor Symposia on Quantitative Biology” took place. It was founded in 1933 by Reginald Harris, the director of CSHL from 1924 to 1936. The Symposia volumes were published by the CSHL. In his introduction to the first volume (Surface Phenomena, 1933), Harris wrote: “The primary motive of the conference symposia is to consider a given biological problem from its chemical, physical and mathematical, as well as from its biological, aspects.” Except for the war period (1943 to 1945), the Symposia took place every year, and the most important advances in biology were announced and debated in these Symposia. The whole history of molecular biology can be found in the series of nine Symposia, beginning in 1941 (Volume IX, Genes and Chromosomes) and ending in 1966 (Volume XXXI, The Genetic Code). The lectures of the 2010 Symposium appeared in volume LXXV (Table 1).

The Phage Group was born at Cold Spring Harbor in 1943. Delbrück defined its mission as “openness … that you tell each other what you are doing and thinking, and that you don't care who has priority” (45). Tempora mutantur! In the summer of 1945, Delbrück started the first of his remarkable series of “phage courses” at Cold Spring Harbor that went on for 26 years. Most of those who participated in them at the beginning (Rollin Hotchkiss, Hermann Kalckar, Leo Szilard, Aaron Novick, Seymour Benzer, Gunther Stent, Giuseppe Bertani, Norton Zinder, Waclaw Szybalski, Frank Stahl, and Bob Edgar, among others) went on to make the most significant contributions on phage or other aspects of molecular biology.

Under Delbrück's influence the group decided to focus its research on a set of T-phages active against the same host, namely, E. coli, strain B. This agreement became known as the “phage treaty of 1944.” One of these phages, T4, was brilliantly exploited by Seymour Benzer (Fig. 6).

Benzer started his scientific career as a solid-state physicist. Influenced by Schrödinger's ideas expressed in What Is Life?, he attended the “phage course” in 1948, after which he decided to work on bacteriophages. Within a few years, he started to investigate a particular mutant of T4 from Hershey's collection, named rII, r signifying rapid lysis. Using the recombination technique, he succeeded in collecting a large number of rII mutants with which he could show that mutations were distributed in many different parts of the gene. The resolving power of the system allowed him to detect mutants distant by a single nucleotide. In order to define the gene functionally, he developed the “cis-trans” complementation test and established the shortest nucleotide stretch that comprised a functional genetic unit, which he named a “cistron” (4, 5). That was the first evidence that the gene is not an indivisible entity, as previously believed. Benzer's accomplishments with the rII system are considered among the most elegant experiments in modern genetics, laying down the groundwork for mutation analysis.

The 1946 Cold Spring Harbor Symposium was the first to be held following a 3-year interruption while the Second World War raged. It was organized by Demerec on “Heredity and Variation in Microorganisms” (Vol. XI) and brought together an international group of scientists working on the genetics of various microorganisms. The Symposium was a real breakthrough in bacterial and phage genetics. It is there that Delbrück and Hershey revealed their discoveries on phage recombination (13, 18) and Joshua Lederberg (Fig. 7), then a Ph.D. student in Tatum's laboratory at Yale, announced that sexual recombination occurs between bacteria, using a new Escherichia coli strain, K-12, which was used for almost all subsequent research in bacterial genetics (25). For recombination experiments, Lederberg used Tatum's nutritionally deficient double mutants and selected for prototrophic recovery. While carrying out these experiments, he was not aware that Tatum's E. coli K-12 was one of the rare strains containing an F plasmid (26).

Lederberg was one of the most creative geneticists. With his wife Esther and with his students N. D. Zinder and M. L. Morse, he discovered extrachromosomal genetic particles, which he called “plasmids,” responsible for the transfer of genetic characters between male and female bacteria. He then discovered a novel way of genetic transfer through bacteriophages, a phenomenon he called “transduction” (34, 47). It is worth mentioning two important technical contributions that he and his wife made: the development of replica plating and the introduction of the chromogenic substrate ONPG (o-nitrophenyl-β-D-galactopyranoside) for β-galactosidase assays.

In 1958, at the age of 32, Lederberg shared the Nobel Prize for Physiology or Medicine with Beadle and Tatum.

André Lwoff, representing the “postwar” Institut Pasteur group, discussed work done during the war—in particular, Jacques Monod's discovery of diauxic growth (32). This marked the beginning of the history of the French school of molecular biology  (see EcoSal Chapter Escherichia coli and the French School of Molecular Biology) [42]). It seems necessary, however, to highlight some important milestones of the postwar period, obtained mainly by the Institut Pasteur group in Paris (André Lwoff, Jacques Monod, and François Jacob [Fig. 8]). It should be remembered that, while bacterial genetics was flowering at Cold Spring Harbor, World War II was devastating Europe and France was occupied by the Germans.

 

GENE REGULATION

Gene regulation started at the Institut Pasteur in Paris with the paradigm of the “Lactose” system. During World War II, the three protagonists who mainly contributed to the development of gene regulation, André Lwoff, Jacques Monod, and François Jacob, were deeply involved in fighting the Nazi occupation. Lwoff's laboratory, in the famous attic, was an active center of the Underground; Monod joined the French Resistance movement, became head of an armed resistance group, and later, following the liberation of Paris, joined the French Army, where he played an important role. After the war ended, Monod joined Lwoff's laboratory, where he had already spent some time during the German occupation.

François Jacob at the beginning of the war 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, he was very severely wounded during the Normandy landing in 1944. Because of his injuries he had to give up the idea of becoming a surgeon. By joining Lwoff's laboratory in 1950, he was given the opportunity to begin research “at the right place and at the right time,” as he recalls.

The discovery of diauxy (33) led Monod to work on β-galactosidase induction and to carry out a genetic analysis of the lactose system. He established that, in E. coli, the synthesis of β-galactosidase depended on three genes: gene z, which governed the capacity or inability to produce the enzyme; gene y, responsible for the synthesis of lactose permease; and a third 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 were closely linked (2).

Around the same time (1949), André Lwoff uncovered the mechanism of lysogeny: 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 he dubbed the prophage, and induction of the prophage (e.g., by UV radiation) would then lead to multiplication and release of the phage (29).

After their arrival at Lwoff's laboratory, Jacob and Wollman, taking advantage of strains isolated by Hayes and Cavalli-Sforza that were capable of transferring genetic material to female bacteria with high frequency (Hfr strains), succeeded in unraveling the mechanism of the sexual process, thus providing a powerful tool to begin studying gene regulation (23). Starting in 1957, a remarkable and uninterrupted series of fundamental experiments led Jacob and Monod to propose a model for the regulation of gene expression (21).

Monod and Jacob first decided to use bacterial conjugation for a genetic analysis of the lactose system. The i, z, and y mutants isolated by Monod were inserted in various combinations into either male or female bacteria. With Arthur Pardee, who was spending a sabbatical year at Institut Pasteur, Jacob and Monod performed one of the most famous experiments in molecular biology, known as the PaJaMo (or PyJaMa) experiment (37); for details, see EcoSal Chapter (Escherichia coli and the French School of Molecular Biology) (42). This experiment, showing that the i⁺ gene is dominant over the i⁻ gene, led to the model of negative regulation: the i⁺ gene produces a factor called "repressor," which blocks the expression of the z⁺ gene. The experiment became also the experimental basis for development of the messenger RNA concept and of the operon model. The operon, defined as a coordinated unit of expression constituted by an operator and a group of structural genes linked to the operator (22), was based essentially on genetic approaches. The chemical nature of the repressor and the direct evidence of rapidly turning over, unstable mRNA remained to be demonstrated.

In their first review on genetic regulation, in 1961, Jacob and Monod proposed that the Lac repressor was RNA (21). If it were true, that would have been the first known regulatory RNA. Thereafter, an increasing amount of genetic evidence accumulated, such as suppressible and thermosensitive mutations, which suggested that the repressor is a protein. Five years later, by isolating and purifying the Lac repressor, Gilbert and Müller-Hill proved that it was indeed a protein (15).

Before direct evidence of the existence of messenger RNA was obtained in 1961 (Fig. 9) (7, 16, 17, 40), the scientific community (re)discovered a puzzling experiment published 5 years earlier by Volkin and Astrachan (43). These authors had found that after a short labeling of E. coli with ³²P during phage T2 infection, a very small fraction of RNA became rapidly labeled even though there was no net synthesis of RNA. Surprisingly, the base composition of this radioactive RNA formed during phage infection corresponded to phage DNA, significantly different from that for E. coli DNA base ratios. Thus, Volkin and Astrachan were the first to discover phage mRNA, different from the host RNA species. One of the direct consequences of the concept of mRNA, followed by its identification, was the breaking and the complete deciphering of the genetic code.

 

STRUCTURE OF DNA AND THE GENETIC CODE

The discovery by Avery et al. (2) that the “transforming principle” was DNA brought to an end the long-lasting dispute on the nature of the gene. Erwin Chargaff reached an important step that led to the understanding of the chemical nature of DNA in 1951 (8, 9) by determining the base composition of DNA from a variety of animal and microbial species. He showed that while the molar ratios of the bases of DNA from different origins varied markedly, those for adenine to thymine and guanine to cytosine remained close to one. These ratios, called “Chargaff's rule,” played an important role in the development of Watson and Crick's elucidation of the structure of DNA in 1953 (Fig. 10) (44).

The elucidation of the structure of DNA was one of the most fundamental and consequential discoveries of 20th century biology because it gave a physical and chemical meaning to the concept of the gene and made possible the analysis of the mechanisms by which DNA was read and genetic information was regulated. Watson and Crick ended their 1953 paper with the famous sentence: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a copying mechanism for the genetic material.” In 1962, Francis Crick, James Watson, and Maurice Wilkins were awarded the Nobel Prize for Physiology or Medicine for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material. It may be of interest to recall that Wilkins had provided to Jim Watson, who was visiting the King's College in London, the X-ray diffraction data that Rosalind Franklin had collected on the B form of DNA. That led him and Crick to the idea that DNA existed as a double helix.

The copying mechanism was established a few years later by two young scientists: Matthew Meselson (Fig. 11), a doctoral candidate, and Frank Stahl (Fig. 12), a postdoctoral fellow (31). According to the Watson and Crick model, several replication mechanisms could be considered: conservative, semiconservative, and dispersed. In an elegant experiment, Meselson and Stahl (Fig. 13) extracted DNA from E. coli cells grown in heavy followed by light nitrogen media and analyzed the differently labeled DNA species by density gradient centrifugation. They proved that DNA replication was semiconservative: when the double-stranded DNA was replicated, the daughter double-stranded helices consisted of one strand coming from the original helix and the other one coming from the newly synthesized strand.

Once the DNA structure was established, the main question that remained to be resolved was how the sequence of nucleotides might code for the sequence of amino acids in a protein. Around the same time, Crick introduced the idea of “central dogma,” stating that genetic information moves from nucleic acid to proteins (what people have called “the Gospel according to St. Francis … Crick”) and no information can get back from protein to nucleic acid (11, 12). The problem of the genetic code was raised. Intellectually, the coding problem had been brought up before, when Frederick Sanger determined the amino acid sequence of bovine insulin and revealed that the amino acid residues are arranged in a definite, genetically determined sequence with no specific rule (38). Therefore, a code was needed to explain the presence of all information in the protein.

In 1961, Brenner (Fig. 14) and Crick designed an elegant experimental strategy (10) to determine the nature of the genetic code using the paradigm of Benzer's rII system. By combining deletion, addition, and frameshift mutations, they were able to generate functional rII gene when groups of 3 plus or 3 minus mutations were brought together. Therefore, before the actual code was known, they could conclude that the translating frame read the code by triplets.

The discovery of metabolically unstable mRNA by Jacob and Monod (21) as an intermediate in information transfer from DNA to protein led to the conclusion that rRNA could not be the template for protein synthesis. This suggested that, if mRNA did carry information needed for the synthesis of specific proteins, then one should be able to reconstitute a cell-free system in which a specific mRNA attached to ribosomes would program the synthesis of the corresponding protein.

In 1961, at the Fifth International Congress of Biochemistry in Moscow, Marshall Nirenberg and Heinrich Matthaei announced that, in a cell-free system from E. coli, a synthetic polynucleotide, poly(U), promoted the formation of a single amino acid polypeptide, polyphenylalanine, thereby breaking the genetic code (36).

The deciphering of the genetic code brought about a fierce competition between the groups of Nirenberg (Fig. 15) and Severo Ochoa. Ochoa had been awarded the Nobel Prize for his discovery, with Marianne Grunberg-Manago, of polynucleotide phosphorylase. This enzyme catalyzes the synthesis of randomly ordered polynucleotides and was at the time used to synthesize these synthetic messengers. The use of a large variety of random polynucleotides that contained different combinations and proportions of bases resulted, between 1961 and 1963, in the determination of the nucleotide composition of about 50 amino acid-specifying codons by the Nirenberg and Ochoa groups (35). During this period, at least 20 papers were published by the two laboratories. Although the two groups had deciphered the nucleotide composition of RNA codons, the nucleotide sequences were unknown.

An important step to determine the nucleotide sequence of the codons was the discovery by Nirenberg’s group that phenylalanine-tRNA was an intermediate in the synthesis of polyphenylalanine directed by poly(U). The next step was the demonstration that poly(U) stimulated the binding of radioactive phenylalanine-tRNA to ribosomes, which prompted Phil Leder and Nirenberg to set up an innovative “Millipore filter” method. The idea was that the filters would retain ternary complexes of radioactive aminoacyl-tRNA-trinucleotide-ribosomes, whereas the radioactive aminoacyl-tRNA would be washed through the filter. The method worked well, and between 1963 and 1965 Leder and Nirenberg published the nucleotide sequences of 54 of the 64 RNA codons (35).

The title of the 1966 Cold Spring Harbor Symposium (Vol. XXXI) was The Genetic Code. By that time the genetic code had been completely deciphered, and it was shown to be universal and degenerate. In 1968 the Nobel Prize in Physiology or Medicine was awarded jointly to Robert W. Holey, Har Gobind Khorana, and Marshall W. Nirenberg “for their interpretation of the genetic code and its function in protein synthesis.”

Crick's “sequence hypothesis” stated that the amino acid sequence of a protein is specified by the nucleotide sequence of the gene determining that protein. It assumed that a correlation had to exist between the order of codons in the gene and the order of the corresponding amino acid in the polypeptide chain (11). The first direct evidence for this colinearity was obtained by Sydney Brenner and coworkers (39) by using a class of suppressible amber mutations affecting the head protein of bacteriophage T4. The mutations led to the production of peptide fragments having the same length and occurring in the same order as the segments defined by the genetic map.

A more accurate relationship between DNA base sequence and amino acid sequence was obtained by studies on altered enzymes in bacterial mutants. Charles Yanofsky and coworkers, using the E. coli tryptophan synthetase system, succeeded in determining the colinearity of gene and protein. The genetic maps from a collection of point mutants of the tryptophan synthetase gene were established in parallel with the peptide maps of the corresponding protein. They showed that specific mutational changes at a single site could be related through the code to specific amino acid substitutions (46).

 

RESTRICTION AND MODIFICATION

Escherichia coli and its phages were the prominent actors in the development of recombinant DNA technology, based mainly on the discovery of restriction enzymes.

In the early 1950s, Luria (27) had discovered a phenomenon called phenotypic modification. He observed that a phage that grew well on a given E. coli strain often grew poorly on another E. coli strain, forming only a few plaques. Phage isolated from these plaques grew well on the second strain but lost the ability to grow on the original one. Werner Arber (Fig. 16), in 1965, while studying host-controlled modification of phage lambda, the same phenomenon discovered by Luria, showed that the host, which restricted lambda, had an enzyme that degraded DNA (restriction enzyme). This restriction enzyme did not degrade the DNA if it had been modified by methylation. Thus, the restriction and modification exist as a paired system, the function of which is to protect host DNA but to destroy foreign DNA (1). For the discovery of restriction enzymes, Werner Arber was awarded the Nobel Prize in 1978, shared with Daniel Nathans and Hamilton Smith.

Arber's daughter, Silvia (who is now a professor in Neurobiology at the University of Basel and at the Friedrich Miescher Institute), wrote a composition entitled “The tale of the king and his servants” to explain her father's discovery to her schoolmates:

“When I come to the laboratory of my father, I usually see some plates lying on the tables. These plates contain colonies of bacteria. These colonies remind me of a city with many inhabitants. In each bacterium there is a king. He is very long, but skinny. The king has many servants. These are thick and short, almost like balls. My father calls the king ‘DNA’ and the servants ‘enzymes.’ The king is like a book, in which everything is noted on the work to be done by the servants. For human beings these instructions of the king are a mystery.

My father has discovered a servant who serves as a pair of scissors. If a foreign king invades a bacterium, this servant can cut him in small fragments, but he does not do any harm to his own king.

Clever people use the servant with the scissors to find out the secrets of the kings. To do so, they collect many servants with scissors and put them onto a king, so that the king is cut into pieces. With the resulting little pieces it is much easier to investigate the secrets. For this reason my father received the Nobel Prize for the discovery of the servant with the scissors.”

In 1997, Fred Blattner and his group published the complete genome sequence of Escherichia coli K-12 (Fig. 17) (6). Thus came to an end the first chapter of the extraordinary saga of an organism that for many decades played a central role in the “golden age” of molecular biology. What the next chapter will reveal cannot even be guessed.