An Interview with Manuel and Ann Varela: Salvador Luria and Viral Replication—Understanding it; Preventing it?

Nov 17, 2020 by

1) Salvador Luria—Born in Turin, Italy—how did he make his way to the U.S., and where did he first begin his studies?

Dr. Salvador Luria was a Nobel Laureate and molecular biologist extraordinaire. He would become famous for his pioneering discoveries on bacteriophage genome structure and their mode of viral replication. Salvador Edward Luria was born on the 13th of August, in 1912, in the Northern Italian town of Turin. His parents were Davide, an accountant, and Ester Luria. He had an older brother.

His teachers heavily influenced Luria as a young boy growing up in the escalation of fascism in Italy. Luria, like them, had opposed the political movement.

Luria excelled in mathematics and language in his pre-college courses. His admitted lack of self-motivation caused his grades in chemistry and biology to be subpar. He claimed to have chosen to study pre-medicine and pursue medicine as a career goal due to his parents’ insistence, not personal preferences.

2) Where did Luria get his medical training?

Luria attended the University of Turin medical school and, while a medical student, studied under the supervision of Professor Giuseppe Levi. The latter was a noted mentor of future prominent scientists, such as Rita Levi Montalcini and Renato Dulbecco. Luria graduated in 1935 and earned his M.D. summa cum laude. No master or Ph.D. degrees were formally given, as the program was highly selective in the Italian higher educational system, and only the highest achieving students were granted those degrees.

For the next year, Luria was enlisted in the Italian army, as military service was compulsory. He served as a medical officer and completed courses in radiation biology at the University of Rome. This placement provided Luria with an introduction to Max Delbrück’s genetic theories. He also met Enrico Fermi, the famous physicist. About Max Delbrück’s ideas regarding the gene as a molecule, Luria wrote later; they seemed to “open the way to the Holy Grail of biophysics.”

In 1938, Luria was hopeful to work with Delbrück in the United States after receiving a fellowship. Still, human beings of Jewish descent were barred from Benito Mussolini’s fascist administration’s academic research fellowships. Luria then fled to Paris, France, and remained there until 1940 when the Nazi German armies invaded. While in France, he attended the Institute of Radium.

Incredibly, it has been reported that Luria rode a bicycle, almost 500 miles (about 804 kilometers), to Marseille and obtained an immigration visa to the United States in June 1940.

Once safe in New York City, Luria changed his name from Salvatore to Salvador. A Rockefeller Foundation fellowship at Columbia University’s College of Physicians and Surgeons was granted to Luria in 1940 with Fermi’s assistance. Luria took Zella Hurwitz, Ph.D., to be his bride in 1945. She was a Professor of Psychology at Tufts University in Massachusetts. The couple has a son named Daniel. Luria was, alas, a Research Assistant in Surgical Bacteriology. At Cold Spring Harbor Laboratory, see Figure 43, Luria collaborated with Max Delbrück and Alfred Hershey.

File:Novick Stocker Papazian EMLederberg Luria Lindegren.jpg

https://en.wikipedia.org/wiki/File:Novick_Stocker_Papazian_EMLederberg_Luria_Lindegren.jpg

Figure 43. A photo of Aaron Novick, Bruce A. D. Stocker, Haig Papazian, Esther M. Zimmer Lederberg, Salvador Luria and Geraldine Lindegren at the Cold Spring Harbor Symposium of 1953.

Luria’s next move was to the University of Illinois at Urbana-Champaign. In his early years there, he and Giuseppe Bertani discovered the phenomenon of host-controlled restriction and modification of a bacterial virus.

Luria started as an Instructor and continued up the ranks to become an Associate Professor at Indiana University from 1943 to 1950. Luria’s first graduate student was none other than James D. Watson, who, along with Francis Crick, discovered the structure of DNA. Interestingly, in 1953, Luria was denied a passport and barred from presenting a paper at a scientific conference in Oxford, England, because of his political activism. Thus, the responsibility would go to a young Jim Watson to present the latest work from the American “Phage Group” to the audience in attendance. The scientific investigators who were members of the pioneering “Phage Group” were convinced that the true nature of the gene could be elucidated by the study of viruses that infected bacteria. Watson thus presented Alfred Hershey’s famous Chase-Hershey experiment conveying the fact that DNA was the hereditary material.

3) Luria shared the Nobel Prize with Max Delbrück and Alfred Hershey—what were their singular discoveries?

Max Delbrück, Alfred Hershey, and Salvador Luria would share the medicine or physiology Nobel Prize in 1969 for their discoveries regarding the viral genetic structure and their replicative nature. See Figure 44. Typically, viral genomes are enclosed in a protein shell called a capsid. Occasionally, viruses harbor a biological membrane called an envelope. Together, Delbrück, Hershey, and Luria had discovered that the gene structure of viruses was of a chemical nature, namely, nucleic acids, such as RNA or DNA. They also learned how to determine the numbers of phages that burst open a single bacterial cell. They further found that the genomes of phages exchanged genetic information with one another. In particular, Delbrück and Luria would provide shattering data supporting the idea that evolution was driven by natural selection of randomly produced spontaneous genetic mutations.

File:Virus structure simple.png

https://commons.wikimedia.org/wiki/File:Virus_structure_simple.png

Figure 44. Simplified diagram of the structure of viruses.

Other notable Phage Group participants include Jim Watson, Francis Crick, Seymour Benzer, Sydney Brenner, and Matthew Meselson. Like these Phage Group colleagues, Delbrück, Hershey, and Luria are considered early forerunners of molecular biology.

Luria worked in Max Delbrück’s lab at Vanderbilt University. Delbrück is covered elsewhere in this book. Delbrück was a key participant with Emory Leon Ellis in performing the famous “one-step” growth curve experiment using bacteriophage viruses. In 1939, Ellis and Delbrück determined the number of phages released from the cell of a single bacterium after bursting it open—the so-called “burst size.” Delbrück also worked with Salvador Luria in the early 1940s to determine the mechanism for bacterial resistance to phage infection. They discovered that mutation played a role in phage resistance. Delbrück’s work helped to definitively demonstrate that mutations occurred spontaneously in a random fashion rather than by induction. Working with W. T. Bailey, Jr., Delbrück was involved in the experiments designed to show that phage genome recombination occurred in bacteria. These works would play a large role in the garnering of the Nobel for him. Later, Delbrück moved on to study fungi and their response to light sources.

The life and science of Alfred Hershey is the focus of a chapter in our 2018 book titled “The Inventions and Discoveries of the World’s Most Famous Scientists.” Hershey is most famous for his work with Martha Chase. They discovered that the genetic material that entered the bacterium host was DNA, rather than protein like most other investigators had fully expected. Thus, the Chase-Hershey data suggested that the gene’s molecular disposition was biochemical and constituted nucleic acid. This discovery alone was instrumental in enhancing the focus towards the elucidation of the DNA structure by Watson and Crick and, to no small extent, those of Maurice Wilkins, Rosalind Franklin, and Raymond Gosling.

As mentioned above, Salvador Luria and Max Delbrück discovered that bacteria acquired resistance to phage infection through spontaneous genetic mutation and not necessarily by adaptation in the Lamarckian sense. Their famous fluctuation test was legendary. It is still widely presented in textbooks dealing with genetics, virology, and molecular biology. Students in various laboratory courses are taught these concepts by repeating the famous experiment themselves. Luria would also become famous for demonstrating that mutation occurs in bacteriophages’ genomes, as had previously been observed with bacterial host mutation. Luria also performed classic experiments dealing with lysogeny and transduction.

In the lytic cycle, the bacterium is killed by exploding it, a process called lysis. Lysogeny is a process in which the host cell remains intact once a virus enters a prophage stage. During the prophage phase, the viral genome integrates into the bacterial genome. The host bacterium, now considered a lysogen in this state, can grow like a common bacterium via binary fission. Transduction involves the transfer of genetic material to bacteria by using a phage as the delivery vector. Luria’s studies in these latter areas were critical to the advancement of molecular biology. Investigators would use Luria’s discoveries to learn about, for example, regulation of gene expression, DNA cloning, and transfer of genetic programming to different species and future generations.

4) One of Luria’s most well-known discoveries was that bacteria mutated spontaneously into “phage resistant” forms. Why is this important?

Luria became famous worldwide amongst bacterial geneticists and later among molecular biologists for his brilliant discovery on the mechanism of bacterial resistance to phage infection. The celebrated experiment conducted by Salvador Luria and Max Delbrück was a mastermind in its design. The story of its conception was legendary, and the experimental scheme was pure genius. What is more, the experiment was founded on the workings of a gambling slot machine!

Working with Delbrück, Luria tested the controversial subject of phage resistant bacteria and its molecular, cellular mechanism. Two conflicting ideas had emerged to explain how a bacterium could develop recalcitrance to lysis by a lytic phage. One idea, called “mutation,” which had been invoked to explain phage resistance, was that the immunity resulted from selecting randomly existing spontaneous mutants in a bacterial population. The second idea, called “acquired hereditary immunity” for phage resistance in the bacteria, held that the phage triggered immunity by a process reminiscent of a Lamarckian mode of evolution.

The formulation for the virtuoso experiment started shortly after Luria’s arrival to Bloomington, Indiana, to begin a new post as a young professor. Luria attended a faculty dance, and he encountered an unnamed colleague playing a slot machine. Luria had chided the gambling colleague, who was focused on the jackpot. Suddenly, Luria realized that mutating bacteria could be envisaged as a sort of a bank bonanza. He reasoned that such jackpots in a slot machine obeyed a Poisson distribution, describing a series of random events considering rare bonanza outcomes.

Luria hypothesized that bacterial phage resistance manifested itself by selecting rare and random spontaneously occurring mutants—the bonanza. These random mutants could appear in a series of “jackpots” in culture—the Poisson distribution. In other words, the selection of random spontaneous mutations was analogous to the occurrence of rare jackpots, conferring a desirable genetic program capable of possessing a useful phenotype.

On the other hand, if phage induction were responsible for bacterial resistance to phage infection, then the jackpot mutants would be evenly distributed in a bacterial culture. In either of the two cases, the average mutant numbers would be similar. However, the distribution would differ depending on whether random spontaneous mutation or phage induction of resistance was at play. One merely had to test the idea in the laboratory! One grand experiment could discern between the two conflicting hypotheses: the spontaneous bacterial mutant selection hypothesis (mutation) versus the bacterial mutation by the phage induction hypothesis (“acquired hereditary immunity”).

The experiment would become known as the fluctuation experiment. Luria and Delbrück used the bacteriophage called alpha (α) and its dedicated bacterial host, Escherichia coli strain B, as their laboratory test subjects. They cultured the Escherichia coli cells that were sensitive to phage α. Then they used the Escherichia coli from the culture to inoculate a large flask with broth medium and a series of test tubes with a few milliliters of broth medium, numbering well over 180 cultures! Next, Luria and Delbrück took multiple samples from the large flask and a small representative sample from each of the hundreds of test tubes. They tested all of them for susceptibility to phage α or resistance to the virus.

The startling result was an even distribution of phage α-resistant Escherichia coli mutants from the large flask. On the other hand, there was a distinctly measurable fluctuation in the number of α-phage-resistant Escherichia coli mutants from the hundreds of individual cultures. That is, the number of phage resistant mutants varied widely—it fluctuated. Thus, Luria and Delbrück concluded that the “mutation” hypothesis was correct. That is, the bacteria developed resistance to phage infection by the selection of random and spontaneously occurring mutations.

Their famous experiment validated statistically that inheritance in bacteria follows Darwinian philosophies. Besides, mutant bacteria randomly occurring can continue to bestow viral resistance without the presence of the virus. Luria and Delbrück published their innovative discovery in a 21-page elegantly written tome in the 28th volume of the journal Genetics in November of 1943. The molecular biology world would never be the same. The last vestiges of Lamarckism had been delivered a final blow.

5) True or False: One of Luria’s first graduate students was James Watson—what other famous names did he mentor or supervise?

Indeed, the inimitable James D. Watson was Salvador’s first graduate student at the University of Indiana in Bloomington. Watson had considered himself fortunate that Luria was still a young professor. Watson had all of Luria’s attention and funding. Watson also picked up a kind of brashness that he acquired from Luria’s example. When Luria occasionally heard of an unsound scientific idea, he was quite terse in his immediate dismissal of it. Soon, Watson followed Luria’s lead and would harbor the same irascibly critical manner for the rest of his life.

In graduate school, Watson took Luria’s course in virology despite hearing rumors that he treated his students like dogs. Watson later reported that Luria’s lectures were mesmerizing, hinting that the future wave was to be held in genetics. It was the first time that Watson had ever heard of a virus. Luria had also lectured on his exciting work with Delbrück. They measured a single phage’s ability to infect a single bacterial cell and use it to produce hundreds of identical progeny phages.

Under Luria, Watson’s Ph.D. thesis involved determining the effects of X-rays on phages’ ability to undergo recombination and produce viable phage progeny within the bacteria. Luria suggested that Watson evaluate the extent to which X-rays genetically damaged the phage’s multiplicity of reactivation system. It was a phenomenon based on a discovery by Luria and Delbrück that phages that were inactivated by X-rays could nevertheless produce active phage progeny. Two phages are genetically recombined when inside a bacterium. The internal recombination would presumably create an active bacteria-lysing phage. Watson started the studies based on Luria’s initial ideas. Soon Watson became more independent and finished the project to everyone’s satisfaction. At 22 years of age, Watson successfully acquired his Ph.D. from the University of Indiana under Luria’s close guidance in 1950.

Dr. Renato Dulbecco was a postdoctoral fellow in Luria’s lab, starting in 1947. Interestingly, Luria’s United States citizenship transpired in 1947. Meanwhile, under Luria’s guidance, Dulbecco learned valuable virological-based laboratory techniques, which would help him in later years to study cancer and win a Nobel Prize in 1975.

In 1949, Dr. Giuseppe Bertani would study lysogeny of Escherichia coli and Shigella bacteria by phages called P1, P2, and P3, in Luria’s laboratory. Luria and Bertani developed a culture medium, which they called “lysogeny broth,” which later was called “Luria broth,” and later Luria-Bertani (LB) medium. See Figure 45.

File:Laboratoorne sööde "LB" ehk "lysogeny broth" on sobiv bakterite kasvatamiseks..JPG

https://commons.wikimedia.org/wiki/File:Laboratoorne_s%C3%B6%C3%B6de_%22LB%22_ehk_%22lysogeny_broth%22_on_sobiv_bakterite_kasvatamiseks..JPG

Figure 45. One of the most common laboratory media – LB, lysogenic broth, known later as Luria-Bertani broth.

Today, the famous LB medium is widely used to cultivate many bacterial species in molecular biological laboratories around the world. Genetic cloning in molecular biology labs frequently involves transformation into Escherichia coli host cells and growth on LB agar Petri plates. See Figure 46.

File:E. coli plate.JPG

https://commons.wikimedia.org/wiki/File:E._coli_plate.JPG

Figure 46. This is a photo of a Luria Bertani agar plate streaked with Escherichia coli, forming colonies.

6) Some of Luria’s later work was at MIT (Massachusetts Institute of Technology). Here he investigated cell membranes—what did he find?

By 1959, Luria became the Microbiology department chair at the Massachusetts Institute of Technology (MIT). His new emphasis in research was on cell membranes and bacteriocins.

A sabbatical to Paris allowed him to study at the Pasteur Institute in 1963. His discovery of bacteriocins effects on the function of cell membranes encouraged him to continue his research at MIT. He found that holes in the cell membrane played a critical role in cell membrane function. Luria was named Sedgwick Professor of Biology at MIT in 1964. In 1965, Luria was named a non-resident Fellow at the Salk Institute for Biological Studies. In 1970, Luria was appointed Institute Professor at the Department of Biology at MIT. In 1972, Luria became chair of the Center for Cancer Research at MIT.

In Luria’s time, the bacteriocins were called colicins. Luria studied those he named “E1” and “K.” Dr. Kay Fields and Luria discovered that the bacteriocins prevented active transport of sugars into Escherichia coli, which then starved to death. The microbes could not undergo glycolysis. In another study, Dr. David Feingold and Luria discovered the bacteriocin E1 enhanced the uptake of hydrogen ions, which are positively charged ions known as cations. The increased proton permeability led to the collapse of the so-called proton motive force, which then prevented ATP production, leading to the killing of the target cell.

Today more than 20 bacteriocins reside in Escherichia coli, and they are found in many other bacterial species. The bacteriocins inhibit the growth of competitor bacteria in several ways. As Luria discovered, one method of bacterial growth inhibition from the bacteriocins involved their action on target membranes. Luria and colleagues found that these bacteriocins inhibit active transport of nutrients into target bacteria or enhance the entry of unwanted ions into the bacteria. Either of these outcomes, i.e., nutrient entry prevention or ion entry enhancement, is detrimental to the bacterium. It can die from either of these phenomena. The bacteriocin manufacture in bacterial producers is directed by plasmids known as bacteriocinogens. Typically, UV light radiation can stimulate the bacteriocinogens into action, causing the release from producers of the bacteriocins. The released bacteriocins then migrate to their unsuspecting target bacteria and kill them.

Target bacteria, however, can fight back against the bacteriocins. There are several bacteriocin resistance mechanisms. One scheme involves reducing bacteriocin binding or membrane insertion. Another tool sequesters the bacteriocins, making them unavailable for antibacterial action. A third system uses an efflux pump or a bacteriocin transporter to expel the bacteriocins from the bacterium. Lastly, the target bacterium can simply digest the bacteriocins into inactive forms.

7) In his later life— Luria enjoyed reading and studying the humanities—and several other realms. Can you tell us a few?

The assorted humanities indeed deeply enamored Luria. Luria had inspired graduate students to read on topics that were not necessarily related to the hard sciences. Interestingly, Luria even gave periodic lectures on world literature. After his retirement, Luria penned several reflective essays and prepared popular public addresses related to the humanities. He wrote a compelling autobiographical memoir, titled “A Slot Machine, a Broken Test Tube: An Autobiography” in 1984. He had written that a laboratory mishap, a broken test tube, later serendipitously led to the discovery of restriction enzymes, which led to the development of molecular cloning. The story goes that in 1952 at the University of Illinois, Luria and his graduate student, Mary Human, studied DNA lysis in Escherichia coli infected with a phage. When they broke a test tube laden with phage-infected Escherichia coli, they switched to the study of Shigella bacteria. The serendipitous switch in bacteria by Human and Luria led to a surprising new observation. The Escherichia coli-specific phages could not replicate when exposed to the Shigella bacteria! The phage infection inhibition in Escherichia coli did not occur if exposed to any other bacteria tested. So, they began a systematic examination of various phages and bacteria hosts to reproduce the Shigella inhibitory effects of phage replication in Escherichia coli. They noticed that the phage seemed to disappear from Escherichia coli when mixed with a different bacterial species and reappear in the next generation of the same Escherichia coli host cells.

Human and Luria referred to the process as a host-induced mechanism and later as a restriction-modification system. Follow-up studies of the disappearance-reappearance phenomena by E.S. Anderson, Giuseppe Bertani, and Jean Weigle suggested that the bacteria were somehow responsible for the restriction-modification process. In 1962, Daisy Dussoix, then a graduate student, and her advisor, Werner Arber, postulated that bacterial restriction endonucleases were responsible for the disappearing lambda phage DNA. The new work led to discovering additional restriction enzymes, which could be used for cloning genes!

In his later years, Dr. Luria took the opportunity to advocate for the merit of scientific progress in democratic societal locales. Luria had gone before Senate and Congressional committees and testified on issues important to scientific policymaking. Luria was a prolific writer of scientific papers (over 150 articles), plus essays and four books. He authored a popular award-winning science book called Life: The Unfinished Experiment in 1973, earning the National Book Award in 1974.

In 1955 Luria became an editor for Virology, a prestigious journal, and held the post until 1972. Luria would serve on the editorial boards for the Journal of Bacteriology, Experimental Cell Research, and the prestigious Journal of Molecular Biology. Luria wrote a textbook about viruses called General Virology, publishing the first of three editions in 1953. Virologists considered the book a gold standard.

8) Where did Luria die, and what would you say were his most outstanding contributions?

Luria died on February 6, 1991, at the age of 78 of a heart attack while in Lexington, Massachusetts. Dr. Luria’s most significant scientific contribution would have to be his determination of phage replication mode involving the lytic and lysogenic cycles. See Figure 47. During the lytic cycle, the phage infection results in the lysis of bacterium and the release of hundreds of progeny phages, diffusing to bind other cells and starting the process again.

Each of these steps represents good molecular and cellular targets for modulation. The viral replication stages and the cellular machinery absconded by the viruses are keenly sought after by medical virologists to inhibit them. Hence, viral infection can be thwarted by chemotherapeutics to prevent and treat illnesses produced by viruses, such as infectious diseases and cancer.

In the lysogenic cycle, the bacterium remains alive, and the phage enters a prophage state. As a prophage, the virus’s nucleic acid genome integrates into the genome of the host bacterium. It permits the host to grow via binary fission. The bacterium becomes a lysogen and can remain as such until the prophage is induced by, let us say UV light exposure. The lysogen will consequently enter the lytic stage to produce new progeny and cause the host’s bursting. Luria and Delbrück were the first investigators to determine how many phages were released upon bacterial bursting. The process became known as the determination of the burst size.

File:Figure 21 02 03.png

https://commons.wikimedia.org/wiki/File:Figure_21_02_03.png

Figure 47. Lytic vs. Lysogenic cycles of phage infection in bacteria.

Another outstanding contribution was Luria’s visualization of phages using the electron microscope. See Figure 48. Luria had collaborated with Delbrück and Thomas Anderson to acquire some of the world’s first images of bacteriophages using the then-new electron microscope.

File:ToBRFV Particles02.jpg

https://commons.wikimedia.org/wiki/File:ToBRFV_Particles02.jpg

Figure 48. Electron micrograph illustration of viral particles.

Luria would be bestowed with many accolades during his lifetime. Professor Luria was also a member of the American Academy of Arts and Sciences in 1959, the National Academy of Sciences in 1960, plus the American Philosophical Society in 1964. He was awarded the Lenghi Prize and became a member of the National Academy of Science (Italy) in 1965. In 1969, Luria took the Nobel Prize in Physiology or Medicine, along with Max Delbrück and Alfred Hershey. They clarified the viral replication mechanism and the genetic structure of bacteriophage viruses. Luria earned the Louisa Gross Horwitz Prize, given by Columbia University, in 1969, and was awarded membership to the Institute of Medicine in 1971. As mentioned above, Luria was given the prestigious National Book Award for Life: The Unfinished Experiment in 1974. Luria earned the National Medal of Science in 1991.

For additional information and a list of Dr. Salvador Luria’s most influential scientific publications, visit:

Print Friendly, PDF & Email

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.