An Interview with Manuel F. Varela and Ann F. Varela: Susan Gottesman: Regulating genes, destroying proteins, and reading Microbe Hunters

Oct 14, 2021 by

Microbe Hunters captured my imagination.”

—Susan Gottesman

Michael F. Shaughnessy

1) Susan Gottesman is a well-known microbiologist. When and where was she born, and where did she go to school?

Gottesman made her entrance into the world on May 19, 1945, in Manhattan, New York. Her father and mother, Dorothy (Altman) Kemilhor, earned college degrees in accounting and education. After teaching typing and stenography, her mother became a high school guidance counselor, and her father managed a company that assembled small appliances, such as rotisseries. The family moved to Long Island when Gottesman was about five or six years old.

Gottesman was an avid reader, and her father gifted her a copy of Paul de Kruif’s book, Microbe Hunters, when she was in elementary school. This book was her springboard into the world of science, nature, and research. Her love of reading played a significant role in her future scientific endeavors. Indeed, she participated in a science project in junior high on “Microbe Moe,” which was concerned with how bacteria enter and leave the body. Soon after, a fantastic opportunity to participate in a summer science program located on Long Island presented itself. Halfway through high school, she discovered that N.S.F. summer programs were offered through the Waldemar Medical Research Foundation and were meant for high school students interested in biology. Gottesman applied and got into that program, which she attended for two summers.

Part of the program involved advanced lectures by highly qualified high school teachers from around the area that came in and talked about the discovery of DNA and what it did, which her high school biology course had not covered. Gottesman was attracted to the puzzle-solving at the bacterial level.

Another part of the summer science program involved doing projects. During the first summer, she experimented with mice but was not excited about it. During the second summer, she tried to get bacteria to grow on a defined medium. Gottesman said that the experiments were not sophisticated in retrospect but strengthened her curiosities.

Gottesman graduated magna cum laude from Radcliffe College with her B.A. in Biochemical Sciences. She received her Ph.D. from the Department of Microbiology at Harvard University and became a postdoctoral fellow at the N.I.H. Later, Gottesman went to the Massachusetts Institute of Technology (M.I.T.) as a research associate. She returned to N.I.H. in 1976 as a senior investigator in the Laboratory of Molecular Biology, where she has remained as co-chief of the Laboratory of Molecular Biology, NCI, and an N.I.H. Distinguished Investigator. In 1998, she was elected to the National Academy of Sciences and the American Academy of Arts and Sciences in 1999. She received the Abbott-ASM Lifetime Achievement Award in 2011.

2) Legend has it that Gottesman read the book “Microbe Hunters” when in the 5th or 6th grade—and this set her on her trajectory. Is there any truth to this?

As we briefly mentioned above, indeed, there is much truth to the story. In several interviews and as revealed in a book on Women in Microbiology, published in 2018 by the American Society for Microbiology Press, Gottesman herself related the story about Paul de Kruif’s 1926 Microbe Hunters. The book, an inspiration to generations of scientists, had been a gift from one of her parents, who had routinely given Gottesman books of interest when she was in grade school. Another book that was of significant interest to Gottesman was The Count of Monte Cristo. As a child, Gottesman was a self-described book aficionado and a voracious reader. Gottesman was also mesmerized by the Nancy Drew mysteries. Solving puzzles was a particularly appealing endeavor for her.

In Microbe Hunters, Gottesman spoke of how she had become fascinated with how the scientific process was done as described in the book. In the book, de Kruif’s writing is engaging and dramatic in its scope. The book devotes each chapter to a principal investigator, each devoted to a particular microbe or a microbial phenomenon. The book portrays the various scientists as sort of like detectives solving one mystery after another. The logical reasoning utilized to learn science by using microbes and doing experiments in the laboratory, as described in Microbe Hunters, captivated Gottesman.

Perhaps the most famous microbiologist depicted in the legendary book is Louis Pasteur, who had two chapters devoted to him. One chapter dramatically described how Pasteur solved the mystery of the “mad dogs” who had the dreaded rabies contagion and whose bites could render their hapless victims a much guaranteed slow and painful death but not after becoming insane first. Nevertheless, Pasteur solved the mystery in his laboratory by conducting systematic experimentation on rabid dogs, culturing rabies on spinal cords hanging in glass jars, and using the material to produce a life-saving rabies vaccine.

These depictions of how science was done, logically and experimentally, figuring out mysteries, appealed to Gottesman. Gottesman had become inspired by de Kruif’s famous book to become a scientist. Microbe Hunters changed the course of her life early on. In particular, Gottesman was enchanted with the scientific logic needed to entirely understand how bacteria caused infectious diseases. The book had become a life-long basis for Gottesman’s keen interest in microbiology, the study of microbes. She had maintained an interest in all things microbiological all through primary and secondary schools. During one summer in high school, Gottesman participated in a program devoted to encouraging young people to enter the sciences, and she gained experience working with DNA in the laboratory. The manipulation of DNA in a laboratory setting requires a fundamental knowledge of microbiology. The inspiration of Gottesman by de Kruif and his book lasted well into her college career.

3) Gottesman attended Harvard and was involved in some research there—what was she involved with while there?

Gottesman was an undergraduate student concentrating in biochemical sciences in the late 1960s at Harvard’s Radcliffe College, for women only. Gottesman took a course called Freshman Seminar, which at the time was geared towards bacterial genetics. When Gottesman was at Radcliffe, its president was Dr. Mary Bunting, a bacterial geneticist who studied a bacterium called Serratia marcescens. The microbe produces a bright red pigment on culture media in Petri plates. See Figure 1.

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Figure 1. Serratia marcescens bacterium.

In Bunting’s Radcliffe laboratory, Gottesman spent some time where she gained experience working with bacteria in the research laboratory setting. Presumably, in Bunting’s laboratory, Gottesman gained a first-hand familiarity with preparing fresh culture media by autoclaving, inoculating the media with bacteria, growing cells in broth and solid agar media in incubators, and examining the colony phenotypes, whether red and wild-type or white and mutant. The new lab experience was one of Gottesman’s first encounters with the field of experimental bacteriology, and it solidified her life-long interest in the topic.

Gottesman spent her junior year at Radcliffe as a laboratory technician in the laboratory of Jim Watson, a Nobel Laureate who shared the 1962 Prize in physiology or medicine with Maurice Wilkins and Francis Crick for solving the DNA structure. In Watson’s Harvard laboratory, Gottesman prepared trichloroacetic acid by precipitation. In the biological sciences laboratory of Watson, high tea was served every afternoon, a British practice that Watson had learned from his time spent in England. Attending Watson’s afternoon tea sessions regularly, Gottesman made friends with Gary Gussin, a graduate student studying tRNA molecules. Gottesman would meet other notable young scientists, like Joan Argetsinger Steitz, famous for her work on RNA, and Mario Capecchi, who would later garner a Nobel for his discoveries about gene targeting approaches using stem cells.

Gottesman’s tutor (sort of like an academic advisor) at Radcliffe was Boris Magasanik, an M.I.T. professor and esteemed molecular microbiologist and biochemist. Gottesman and Magasanik met when he had given a guest lecture in her Freshman Seminar course about phase variation. During her senior year at Radcliffe, Gottesman elected to pursue her senior thesis project in Magasanik’s M.I.T. laboratory, where she met Bonnie Tyler, a graduate student conducting her graduate work. Tyler would become a mentor to Gottesman and provide a role model for her and teach her the valuable technique for measuring the kinetics of the β-galactosidase enzyme. The bacterial enzyme cleaves the sugar lactose into glucose and galactose.

Magasanik would become an influential mentor, encouraging Gottesman to study bacterial genetics for her graduate studies. Magasanik said she might consider working in the laboratory of Ethan Singer or Jon Beckwith. She would decide to pursue her interests in bacterial genetics in the laboratory of a young faculty who would one day become one of the world’s most famous bacterial geneticists, Jon Beckwith, at Harvard Medical School in Boston.

4) Gottesman worked with Jon Beckwith. What was her area of investigation, and what did she find?

After graduating from Harvard Radcliffe with her undergraduate degree in biochemistry, Gottesman took the advice of Boris Magasanik and applied to graduate school at Harvard’s medical school in Boston to study under Jon Beckwith. At Harvard’s department of bacteriology and immunology, Beckwith had just started his new research laboratory and had been there just a few years. Beckwith had collaborated with Ethan Signer at the Pasteur Institute in Paris. The duo had discovered how to use transposition to clone genes encoding lactose metabolism machinery from Escherichia coli bacteria elsewhere in the genome near the genetic insertion site for a phage virus called Φ80.

Afterward, before Gottesman had arrived at the Beckwith lab in 1969, two of his postdoctoral fellows, Karen Ippen and Jim Shapiro, had just discovered that they could use the same transposition method of gene jumping to move the genes from bacteria to a phage. The genes encoding a lactose-fermenting enzyme, called β-galactosidase, and a transporter, called permease, were transposed from the Escherichia coli genome into a specific location near the galactose metabolizing genes of the bacterium using the phage lambda (λ) insertion site as a gene marker. At the time, this sort of work garnered plenty of newspaper headlines because it was the first time that any investigator had isolated a known gene, the one for β-galactosidase, from the genome of a bacterium. The identified gene could be visualized as a piece of DNA using an electron microscope. See Figure 2.

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Figure 2. Electron micrograph of a phage λ viral particle, known as the Escherichia virus Lambda species.

When Gottesman entered the Beckwith laboratory in Boston, she got to work as a new graduate student. She invoked the newly established technique of using bacteria and phage viruses to transpose genes from one location of the Escherichia coli genome to another locus. Gottesman had chosen to work with the genes encoding utilization of the amino acid threonine and a lump of sugar called arabinose.

In the Harvard laboratory at Boston, Gottesman used a genetic element called F for the fertility factor, which can insert by conjugation into the bacterial genome to produce an episome, an integrated genetic component. Using this jumping gene system, called F-thr-ara episome, isolated clones of Escherichia coli in which the thr and ara genes moved to another genomic location, in the insertion site for the phage called T1 and near the attachment site of phage Φ80.

Once the F-thr-ara episome had undergone its genome inserting process, another jumping gene phenomenon, Gottesman isolated the new Escherichia coli strain in pure culture. With its new insertion, the F-thr-ara episome, in its new place, she was ready for the next phase of her Ph.D. project. Next, Gottesman used the newly acquired bacterial strain to isolate a transducing-defective phage Φ80 strain that harbored the arabinose metabolizing machinery. The new directed-transposition method showed great promise in generating phages with desirable genes, like sugar metabolizing enzymes. It would prove to be a valuable genetic engineering tool for molecular biologists. The new finding would permit the study of the gene regulatory systems involved in arabinose metabolism.

Gottesman’s work in Beckwith’s laboratory would start a revolution in our understanding of gene regulation. Afterward, it would be discovered that the Ara system, an operon that controlled arabinose metabolism that Gottesman studied, was regulated positively, with metabolizing genes turned on in the presence of arabinose sugar. See Figure 3. A negative regulation mechanism would be found, too. It involved DNA looping around to prevent RNA polymerase from having access to its transcription promoter.

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Figure 3. Model of the chemical structure of arabinose.

5) Gottesman seems to have made her career in the area of biological regulation and enzymes. What is going on in the human cell?

Gottesman conducted several significant studies on the arabinose operon using λ phage early in her career in graduate school. Her following investigations would eventually relate to the biological regulation of enzymes in human cells from these early studies. Let us trace this pathway from phages to humans.

After graduate school, Gottesman became a postdoctoral fellow at the N.I.H., working under Max Gottesman (no relation) in Bethesda, Maryland. At the N.I.H., she attended periodic “Lambda Lunches,” which were seminars that addressed the latest issues on gene regulation. In the meantime, Gottesman performed new experiments that dealt with the stability of two phage λ proteins called N and cII. She discovered that a mutation in the gene called ion (sometimes called the Ion gene), which was located on the Escherichia coli genome, played a part in the control of the N and cII protein stabilities of phage λ. Gottesman had determined that when the Escherichia coli ion gene is defective by mutation, the bacteria degrade proteins.

Among the many effects observed in the mutant ion strains of Escherichia coli, one was the accumulation of high levels for a protein called SulA that resulted after exposure to ultraviolet light. Gottesman referred to SulA as an ultraviolet light-induced cell division inhibitor. The Escherichia coli cells with a defective ion gene had several features, such as making an overabundance of capsule sugars, an inability to become lysogenic by phage λ infection, and a reduced ability to degrade abnormal bacterial proteins.

Next, Gottesman discovered that the Ion protein used ATP energy to undergo its protein degradation process, a function that she called ATP-dependent proteolysis. Interestingly, she discovered that one of the targets for the Ion protein was SulA itself. Thus, the Ion protein functioned to degrade SulA, thereby prevent it from inhibiting bacterial cell division. The new work involving ATP-dependent proteolysis systems would lead Gottesman to make one of the most important discoveries of her career: the small RNA molecules and their effects on post-transcriptional regulation of gene expression. Similar regulatory mechanisms would be discovered working in eukaryotic cells, including those of humans. It seems that the role of small RNAs extends to all types of living cells. These small regulatory RNAs would be a discovery that would place Gottesman forever in the textbooks dealing with molecular biology at all levels of life, including regulation of gene expression systems in humans.

6) Small RNAs and ATP-dependent proteases—why are these things important?

Gottesman is perhaps most well known for her studies discovering the regulatory small RNA molecules, also known as sRNAs, microRNAs, or miRNAs. In specific cases, they are also called riboswitches. These small RNA pieces function in two ways. One mechanism of action is binding to messenger RNA (mRNA) to regulate their protein output by controlling translation in both prokaryotic and eukaryotic cells. The small RNA molecules can turn on or off translation, thus increasing or restricting the protein amount that a cell produces. Further, the small RNAs can control the degradation of messenger RNA by reducing or enhancing the availability of the genetic messages, affecting protein production.

The second mechanism of small RNA action is to bind proteins directly and influence their activities. The small RNA molecules are known to bind, for example, CsrA, which regulates translation. The complexes formed by CsrA-sRNAs cannot bind to target mRNA molecules, thus affecting the production of target proteins. These small RNA regulatory systems represent a form of the so-called post-transcriptional control mechanisms. The riboswitches bind small biomolecules, such as metabolites, changing their molecular configurations after the small RNA molecules perform their binding activities.

In particular, the small RNAs carry out a wide range of cellular functions, such as silencing the expression of genes, biochemically methylating bases on DNA, modifying ribosomal and transfer RNA molecules, and influencing the stages of the organism development. These small RNA regulators are relevant in medical diseases like cancer, diabetes, and liver disorders. Furthermore, post-transcriptional regulation by these small RNA molecules has been discovered to play essential roles in the growth of cells, the acquisition of specialization functions in developing cells, and in the body’s immune system to prevent infection and cancer.

In one exciting line of research, Gottesman examined the response that Escherichia coli undertook as it suffered through stressful conditions, such as nutrient starvation, drastic changes in pH, high osmotic pressure, damage to its DNA, or when the bacterium reaches the end of its generational life. One of the first of these critical small RNA molecules to be discovered by Gottesman is called DsrA. In later years, she would recall fondly how its discovery, in the early 1990s, was a particularly gratifying exercise in solving a mystery.

This line of investigation started when Gottesman studied the ability of Escherichia coli to produce a protective capsule. The microbe made the capsule by synthesizing colanic acid, which required several regulatory proteins to do so. When Gottesman and her laboratory workers searched the upstream region of the rcsA gene encoding one of these regulatory proteins called RcsA, the promoter region did not seem to have an active site. The active region of the gene was a mystery. Surprisingly, the gene contained a so-called downstream element, which we know today encodes one of these small RNAs, called DsrA, short for downstream from RcsA.

The DsrA small RNA molecule forms a secondary structure consisting of several so-called hairpin loops. The short RNA sequence will form base pairs upon itself, fashioning a loop resembling a hairpin structure. These RNA hairpin loops were found to activate or inhibit translation, depending on the type of three-dimensional structure formed. One of these hairpins stimulates the translation of the RpoS protein. The RpoS molecule is a transcription factor called sigma-38 that aids RNA polymerase during the end of the bacterium’s 20-minute life. DsrA functions as a positive influence by unwinding one of these inhibitory hairpin loop structures on the small RNAs to permit protein synthesis of RpoS. The newly made RpoS then functions during the stationary growth phase when Escherichia coli has reached the end of its life or is suffering from stress. The DsrA-RpoS system helps the bacteria respond to stress, providing a survival mechanism.

More recent studies started in Gottesman’s laboratory shortly after the turn of the 21st century involved another small RNA molecule called RyhB, which was found during a genome-wide search for other short stretches of non-coding RNA molecules. Gottesman first discovered that the RyhB molecule regulates the production of iron-storage and iron-requiring proteins. Interestingly, the RyhB small RNA was itself regulated by a protein called Fur, for ferric uptake regulator, an iron uptake repressor. The RyhB system provided Escherichia coli with a means for surviving iron starvation. Soon, another protein was found to be involved. The molecule was called Hfq, an RNA chaperone protein required for the actions of RyhB and DsrA. The RNA chaperone Hfq protein, thus, helps small RNAs target specific mRNAs to perform post-transcriptional regulation by influencing the stability of these mRNAs and by regulating protein synthesis.

Gottesman is also quite widely credited for her pioneering studies on ATP-energized protein degradation, a process known as proteolytic destruction, and which is performed by a set of enzymes generally called proteases. She had demonstrated that in bacteria undergoing stress, the protease enzymes use ATP hydrolysis to control the delivery of substrates by using adaptor and anti-adaptor molecules to the proteolytic enzymes.

In one exciting project, Gottesman and colleagues found that when Escherichia coli cells respond to environmental stress or enter the stationary phase of growth, proteins are turned over by degrading them so that the released amino acids can be used to help the bacteria survive during their trying times of trouble. During this bacterial stress survival response, the sigma-S (σS) factor for RNA polymerase regulates the necessary genes for their endurance. The activity of the σS factor is itself regulated by degradation, which is performed by a protease called ClpXP that uses ATP for energy. The ClpX part of the protease performs the regulatory function while the ClpP part performs the actual protein cleaving functions, a process called proteolysis. Gottesman discovered that when a so-called response regulator named RssB was phosphorylated by acetyl phosphate, the RssB-phosphate complex greatly stimulated the σS factor degradation by the ClpXP protease enzyme.

Thus, Gottesman found that the σS factor, RssB, acetyl phosphate, and ClpXP work together in a coordinated fashion using ATP to energize the survival process. These findings were significant because it was demonstrated for the first time that during a stress response, bacteria could fine-tune the degradation process to permit RssB to repeatedly deliver a targeted protein, the σS factor in this case, to the protease enzyme. Studies like these arising out of the Gottesman laboratory would make her famous among microbiologists, biochemists, and molecular and cellular biologists and ensure that her contributions were readily included in the biology textbooks.

7) Escherichia coli cells—why are they important, and what did Gottesman’s work show about these cells?

Gottesman was an astute scientific investigator as she studied biochemistry, physiology, and molecular biology of Escherichia coli. This bacterium is a tremendously important microbe for a variety of reasons. In molecular biological circles, Escherichia coli is one of the most used living vehicles for propagating cloned genes and other genetic elements arising from all other living organisms, including humans. For instance, if an investigator clones an important human gene, the DNA encoding that gene will be cultivated in the famous bacterium. Such scientists will use Escherichia coli to foster the application of the gene or to alter it to study the biological effects of the human gene mutation. Many genes from all walks of life were cloned, grown in Escherichia coli, and sequenced early on. One significant example of this technological application occurred during the human genome project, making it possible to determine the human genome map quickly. Thus, the human genome and the genomes of many other living organisms were dependent upon Escherichia coli for their DNA sequence determinations. Without Escherichia coli, knowing these genomes at the primary molecular levels would have been tremendously difficult, such as their DNA and protein sequences.

The Escherichia coli microbe is greatly important in biotechnology, a field of science devoted to producing products we need, such as diagnosis, forensics, medical therapies, vaccines, biotech products, and many other research applications. Quite often, when these desirable products are developed and produced, Escherichia coli is the one organism that biotechnologists turn to so that the product is made most efficiently. Escherichia coli is extensively used because it is easy to manipulate in the laboratory setting. The microbe is readily cultivated in the lab using simple culture media, glassware, facilities, and essential laboratory equipment like autoclaves, fermenters, and incubators. The ease of Escherichia coli manipulation permits investigators to readily scale up, amplify and isolate the desirable products from large batches of bacterial cultures in the laboratory.

Interestingly, the wild-type Escherichia coli bacterium is undoubtedly the most well-known, if not the best understood, living organism in the history of the scientific world. Investigators know more about the biological workings of Escherichia coli than any other organism known to humankind. While we will never know or understand all of it, we unquestionably know more about Escherichia coli than we know about all other life, including humans—especially humans. The extensive body of knowledge about Escherichia coli makes it one of the most influential living cells we have ever studied. We can use our knowledge of the microbe as a comparative basis for understanding all other life. Perhaps an unknown plant or human gene has a hint of its function stored away in the knowledge base generated by our use of Escherichia coli. The human gene can be compared with Escherichia coli, or the human gene can be cloned and placed in the bacterium to study closely. As we alluded to above, Escherichia coli permits us to learn more about all other life. We will undoubtedly continue to use Escherichia coli as our “laboratory rat” in these ways well into the unforeseeable future.

Because Escherichia coli lives in the guts of all known animals, including humans, its gut presence has had significant implications for all of us. For instance, if Escherichia coli is present anywhere outside of a laboratory, whether pathogenic or harmless, the situation is an unpleasant indicator of fecal contamination. While the wild-type Escherichia coli itself is frequently harmless, the indication of fecal material in any non-lab location is a sign that other infectious disease agents can be present, possibly conveying disease through human populations via fecal transmission.

Lastly, while the Escherichia coli is a non-dangerous, harmless, and perhaps even helpful bacterium living in the gastrointestinal tract of all humans and other animals on Earth, the microbe nevertheless has a few extremely dangerous close relatives. These pathogenic, disease-causing strains of the wild-type Escherichia coli can be highly debilitating, if not deadly. The pathogenic relatives of the Escherichia coli can cause terrible illnesses in the gut, such as gastroenteritis, food poisonings, and even stomach or colon cancers. These few rogue Escherichia coli relatives have given the regular wild-type Escherichia coli a lousy impression. Much attention has been given to biomedical scientific laboratories worldwide to mitigate the pathological and deadly effects of these microbial Escherichia coli pathogens. Biomedical scientists have developed antibiotics in clinical settings and invoked prevention measures outside the clinics to treat and thwart these bacterial pathogens.

Gottesman’s scientific work had definite relevance to many aspects of bacterial physiology, molecular biology, and its biochemistry. Her work has spanned many decades of dedicated research into the workings of Escherichia coli and its response to stress, regulation of gene expression, and the biochemistry of protein degradation. She had discovered that the mechanisms involved in using ATP do undergo proteolysis in Escherichia coli. While studying the protease enzymes involved, Gottesman established the regulatory systems concerned, many of which were relevant to other bacteria, particularly microbial pathogens. These regulatory molecules were relevant to eukaryotic cells, including humans.

Likewise, Gottesman’s discoveries and subsequent investigations dealing with the small RNAs have been tremendously helpful to our understanding of gene regulation in many other species of bacteria, especially regarding the physiology of pathogenic bacteria like Salmonella enterica, Pseudomonas aeruginosa, and Helicobacter pylori. Furthermore, her studies on small RNAs directly relate to human biology, such as the microRNA molecules and their effects on the biogenesis, messenger RNA stabilities, and translational systems used to make proteins. Lastly, Gottesman’s scientific contributions regarding the small RNAs have influenced the study of human medicine, and her work has relevance to applications regarding certain human diseases.

8) These small RNAs seem to be linked to cancer, diabetes, and liver disease. What is the mechanism and or relationship?

Indeed, Gottesman’s studies on small RNA molecules directly relate to many aspects of human biomedicine, especially those dealing with cancer, cardiovascular ailments, stroke, diabetes, infection, and diseases of the liver, kidneys, and nervous system. Gottesman’s small RNA systems are relevant indicators of specific human disease and as a potential for medical therapies and serving as promising targets for modulation.

Regarding cancer, the small RNA molecules are involved in many aspects of their biology. For example, microRNAs play a role in conferring a non-differentiated state in tumors, a critical factor in their cell pathology. These microRNAs interact with a variety of protein translational machinery to regulate protein synthesis levels. See Figure 4. Furthermore, specific small RNAs are dysfunctional and dysregulated in cancer cells. A well-known example of this is the microRNA called miR-1-1 (short for microRNA-1-1), which was demonstrated to control differentiation and proliferation in heart cells. Likewise, the closely related miR-1-2 small RNA was shown to be involved in a similar developmental process in muscle cells. In such healthy cells, these two microRNAs serve as tumor suppressors. However, in tumor cells, the genes encoding these small RNA pieces can be mistargeted by modulators or mutationally altered in such a manner that they now confer oncogenesis to form unwanted tumors. If healthy cells are transformed into tumorigenic ones, then loss of cell specialization and function from the dysregulation of their microRNAs contributes to their new cancerous pathology.

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Figure 4. Interaction of microRNA with protein translation process.

Near the turn of the most recent century, circa 2000, small RNA molecules were discovered to play a role as regulators of type I and type II diabetes. One of the first of these sort of microRNA molecules to be discovered was called miR-375. In type I diabetes, miR-375 is overproduced during gene expression, suppressing insulin secretion when glucose is in the blood. On the other hand, it was demonstrated that when the miR-375 small RNA was inhibited, then insulin production was enhanced. It was later shown that the mi-R375 microRNA mediated its regulatory effects by suppressing pyruvate dehydrogenase, an essential enzyme in glucose metabolism. Additionally, the gene encoding mi-R375 microRNA was experimentally knocked out in mice, and the deletion mutation resulted in the mice being unable to control their blood glucose levels, producing a hyperglycemic effect in the mutant mice.

In another study, mi-R375 microRNA was demonstrated to inhibit myotrophin, a protein that plays a role in protein synthesis in heart cells. Thus, this microRNA molecule seems to have a role in cardiac function and development. The implication is that when present in healthy tissues and allowed to function normally, the small RNA molecules serve to stimulate normal cell and tissue development.

In type II diabetes, the microRNA molecule named mi-R125a was implicated in playing a role. When overproduced in specialized pancreatic cells, called β-acinar cells, this specific small RNA molecule produced the type II form of the diabetic condition in laboratory rats. The cellular defect seemed to involve an impaired ability of the β-acinar cells to proliferate to suitable levels, producing an impaired tissue development condition.

The liver appears to be home to several dozen individual microRNA systems. The biological functions of these microRNAs in the liver appear to involve post-transcriptional regulation of gene expression. The microRNAs also play critical roles in performing healthy liver functioning. However, about two dozen unique small RNA molecules have been dysregulated in several liver diseases, especially those involving viral infection and liver cancer. A series of microRNA molecules are involved in the functions of liver viruses, virally infected liver cells, and the pathology of infectious hepatitis illnesses. A fair number of microRNAs are involved in hepatitis C and B viruses. In both of these viruses, microRNAs control the gene expression of virally encoded genes. They are helping the viruses to maintain their presence in large numbers in the infected liver. Thus, much investigative work is focused on finding new ways to inhibit these microRNA systems in virally infected liver cells.

The microRNAs are aberrantly expressed in liver cancer cells, their production being enhanced or reduced in specific malignant liver cancer cases. When expressed in healthy liver cells, many microRNAs work properly in typical healthy liver cell specialization and liver functioning, such as normal metabolism.

Whether the microRNAs in the liver are upregulated, downregulated, or defective in their function, producing liver disease, these molecules nevertheless serve as valuable targets for healthy modulation and restoration of proper function. Much work is devoted to using these liver microRNA systems for the development of chemotherapies. Furthermore, these liver-specific microRNAs can serve helpful roles in the clinical diagnosis of liver disease.

9) What is Gottesman currently doing and researching?

As of this writing, Professor Gottesman has maintained a vigorous research program devoted to studies of bacterial stress responses, environmental changes that bacteria encounter, gene expression regulation at both transcriptional and post-transcriptional levels, and the regulation by small RNAs of protein stability. One of Dr. Gottesman’s most recent papers was published in a prestigious journal officially known as Proceedings of the National Academy of Sciences. Gottesman is an impressive member of this organization. In the recent 2021 PNAS paper, Gottesman related a discovery on a mechanism for rapidly switching the gene expression statuses in bacteria. In the study, Gottesman and her colleagues used a sophisticated technique called fluorescence-based functional screening to discover new regulator factors that maintain an off switch for the expression of small RNA genes, such as RyhB.

These novel regulators fell into two types. The first type involves those which affect RyhB-specific regulators. One of these new specific regulators is AspX, a small RNA molecule that suppresses the gene expression program of RyhB. This regulation system uses a so-called RNA sponging mechanism, which involves new RNA transcripts that can titrate the small RNAs away from their mRNA targets, similar to a sponge. The sponge RNA systems remove the small regulatory RNAs so that gene expression programs of target genes, such as RyhB or circRNA, are themselves quenched. See Figure 5.

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Figure 5. RNA sponge regulatory system.

The second regulator type discovered by Gottesman consisted of the so-called general regulators, which affect the signaling processes of a variety of small RNA regulator molecules. These general regulators were involved with the RNA chaperone Hfq. They worked together by forming new interactions to regulate bacterial metabolism, respond to environmental stress, and affect infectious disease virulence.

In another study, Gottesman collaborated with another group to study a life-threatening pathogen called multidrug-resistant Klebsiella pneumoniae, known in many circles as a “superbug” because of its virulence in causing severe cases of pneumonia. One approach to treatment is phage therapy, in which specific bacteriophage viruses can target and selectively destroy the bacterial pathogen by cell lysis. However, individual variants can emerge in which phage resistance permits the pathogens to survive killing by phage.

In the 2020 study published in the journal mBio, Gottesman addressed this phage resistance problem in Klebsiella pneumoniae by astutely selecting distinctive for various steps in the phage infection process and combining these phages to undergo a combination treatment strategy. Gottesman and her colleagues developed a strategy to predict and discover the various phage combinations that could be used in any future emergence of phage-resistant Klebsiella pneumoniae mutants. Such a phage-finding strategy could help circumvent the spread of the microbe and migration of bacterial pneumonia through various human populations.

Another recent study, also published by Gottesman in 2020 in the journal called PLoS Genetics, deals with a so-called Rcs phosphorelay system. The bacterial phosphorelay system plays a critical role in permitting Escherichia coli to sense their environments for potentially dangerous growth-inhibiting molecules and quickly respond to them. Gottesman’s laboratory found that IgaA, a membrane-bound protein known to attenuate intracellular growth, turns off the phosphorelay system by interacting with RcsD, a phosphotransfer protein, making multiple contacts between them. The multi-component interaction between IgaA and RscD produced a fine-tuning mechanism for sensing the bacterium’s environment. The phosphorelay modulation by IgaA and RscD provide a sensitive regulatory molecular switch mechanism.

Interestingly, in 2019 Gottesman analyzed the relationship between small RNAs and regulation of the famous Krebs cycle, also recognized by many as the tricarboxylic acid cycle and the citric acid cycle. In one study, the Gottesman laboratory found one of these small RNAs was derived from the so-called 3’-untranslated regions (3’UTRs) of specific mRNA transcripts by cleavage of the target RNA using an endoribonuclease enzyme called RNase E. The RNA cleavage product, a small RNA, was called SdhX. The expression of the SdhX was dependent on the expression of a variety of genes located on a genetic operon called sdhCDAB-sucABCD that encodes enzymes relating to the Krebs cycle. When generated by the RNase E cleavage, SdhX then turns off the expression of the gene called ackA, which encodes acetate kinase that catalyzes the interconversion of acetate and acetyl-phosphate. The metabolic result of this sort of regulation by the SdhX small RNA is the accumulation of the acetyl-phosphate, which can then be used to coordinate the signaling and regulation of metabolic pathways dealing with acetate biochemistry and the activity of the Krebs cycle. See Figure 6.

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Figure 6. The Krebs cycle.

10) What have I neglected to ask?

Dr. Gottesman has been quite successful at garnering coveted academic and institutional positions. For instance, Gottesman is the Head of the Biochemical Genetics division and an N.I.H. Distinguished Investigator at the N.I.H. Center for Cancer Research. At the N.I.H. National Cancer Institute, Gottesman is the current chief of the Laboratory of Molecular Biology. Thus, Gottesman has been a positive role model for young female scientists worldwide.

In the late 1990s, Gottesman was elected to the prestigious National Academy of Sciences. Shortly afterward, in 1999, she was elected as a fellow of the American Academy of the Arts and Sciences, and in 2009, Gottesman became a fellow of the American Society for Microbiology. In 2015, Gottesman was bestowed with the Selman A. Waksman Award for her excellence in microbiological research.

In 2008, Gottesman became the editor-in-chief of Annual Review of Microbiology, a prestigious journal within the microbiological sciences. In this venue, Gottesman has had the opportunity to influence research programs dealing with the significance of bacterial physiology, molecular microbiology, and microbial biochemistry.

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