11.1: Using Microbiology to Discover the Secrets of Life

Learning Objectives

• Describe the discovery of nucleic acid and nucleotides
• Explain the historical experiments that led to the characterization of DNA
• Describe how microbiology and microorganisms have been used to discover the biochemistry of genes

Through the early 20th century, DNA was not yet recognized as the genetic material responsible for heredity, the passage of traits from one generation to the next. In fact, much of the research was dismissed until the mid-20th century. The scientific community believed, incorrectly, that the process of inheritance involved a blending of parental traits that produced an intermediate physical appearance in offspring; this hypothetical process appeared to be correct because of what we know now as continuous variation, which results from the action of many genes to determine a particular characteristic, like human height. Offspring appear to be a “blend” of their parents’ traits when we look at characteristics that exhibit continuous variation. The blending theory of inheritance asserted that the original parental traits were lost or absorbed by the blending in the offspring, but we now know that this is not the case.

Two separate lines of research, begun in the mid to late 1800s, ultimately led to the discovery and characterization of DNA and the foundations of genetics, the science of heredity. These lines of research began to converge in the 1920s, and research using microbial systems ultimately resulted in significant contributions to elucidating the molecular basis of genetics.

Discovery and Characterization of DNA

Modern understanding of DNA has evolved from the discovery of nucleic acid to the development of the double-helix model. In the 1860s, Friedrich Miescher (1844–1895), a physician by profession, was the first person to isolate phosphorus-rich chemicals from leukocytes (white blood cells) from the pus on used bandages from a local surgical clinic. He named these chemicals (which would eventually be known as RNA and DNA) “nuclein” because they were isolated from the nuclei of the cells. His student Richard Altmann (1852–1900) subsequently termed it “nucleic acid” 20 years later when he discovered the acidic nature of nuclein. In the last two decades of the 19th century, German biochemist Albrecht Kossel (1853–1927) isolated and characterized the five different nucleotide bases composing nucleic acid. These are adenine, guanine, cytosine, thymine (in DNA), and uracil (in RNA). Kossell received the Nobel Prize in Physiology or Medicine in 1910 for his work on nucleic acids and for his considerable work on proteins, including the discovery of histidine.

Foundations of Genetics

Despite the discovery of DNA in the late 1800s, scientists did not make the association with heredity for many more decades. To make this connection, scientists, including a number of microbiologists, performed many experiments on plants, animals, and bacteria.

Mendel’s Pea Plants

While Miescher was isolating and discovering DNA in the 1860s, Austrian monk and botanist Johann Gregor Mendel(1822–1884) was experimenting with garden peas, demonstrating and documenting basic patterns of inheritance, now known as Mendel’s laws.

In 1856, Mendel began his decade-long research into inheritance patterns. He used the diploid garden pea, Pisum sativum, as his primary model system because it naturally self-fertilizes and is highly inbred, producing “true-breeding” pea plant lines—plants that always produce offspring that look like the parent. By experimenting with true-breeding pea plants, Mendel avoided the appearance of unexpected traits in offspring that might occur if he used plants that were not true-breeding. Mendel performed hybridizations, which involve mating two true-breeding individuals (P generation) that have different traits, and examined the characteristics of their offspring (first filial generation, F₁) as well as the offspring of self-fertilization of the F₁ generation (second filial generation, F₂) (Figure \(\PageIndex{1}\)).

Figure \(\PageIndex{1}\): In one of his experiments on inheritance patterns, Mendel crossed plants that were true- breeding for violet flower color with plants true-breeding for white flower color (the P generation). The resulting hybrids in the F₁ generation all had violet flowers. In the F₂ generation, approximately three-quarters of the plants had violet flowers, and one-quarter had white flowers.

In 1865, Mendel presented the results of his experiments with nearly 30,000 pea plants to the local natural history society. He demonstrated that traits are transmitted faithfully from parents to offspring independently of other traits. In 1866, he published his work, “Experiments in Plant Hybridization,”¹ in the Proceedings of the Natural History Society of Brünn. Mendel’s work went virtually unnoticed by the scientific community, which believed, incorrectly, in the theory of blending of traits in continuous variation.

He was not recognized for his extraordinary scientific contributions during his lifetime. In fact, it was not until 1900 that his work was rediscovered, reproduced, and revitalized by scientists on the brink of discovering the chromosomal basis of heredity.

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The Chromosomal Theory of Inheritance

Mendel carried out his experiments long before chromosomes were visualized under a microscope. However, with the improvement of microscopic techniques during the late 1800s, cell biologists could stain and visualize subcellular structures with dyes and observe their actions during meiosis. They were able to observe chromosomes replicating, condensing from an amorphous nuclear mass into distinct X-shaped bodies and migrating to separate cellular poles. The speculation that chromosomes might be the key to understanding heredity led several scientists to examine Mendel’s publications and re-evaluate his model in terms of the behavior of chromosomes during mitosis and meiosis.

In 1902, Theodor Boveri (1862–1915) observed that in sea urchins, nuclear components (chromosomes) determined proper embryonic development. That same year, Walter Sutton (1877–1916) observed the separation of chromosomes into daughter cells during meiosis. Together, these observations led to the development of the Chromosomal Theory of Inheritance, which identified chromosomes as the genetic material responsible for Mendelian inheritance.

Despite compelling correlations between the behavior of chromosomes during meiosis and Mendel’s observations, the Chromosomal Theory of Inheritance was proposed long before there was any direct evidence that traits were carried on chromosomes. Thomas Hunt Morgan (1866–1945) and his colleagues spent several years carrying out crosses with the fruit fly, Drosophila melanogaster. They performed meticulous microscopic observations of fly chromosomes and correlated these observations with resulting fly characteristics. Their work provided the first experimental evidence to support the Chromosomal Theory of Inheritance in the early 1900s. In 1915, Morgan and his “Fly Room” colleagues published The Mechanism of Mendelian Heredity, which identified chromosomes as the cellular structures responsible for heredity. For his many significant contributions to genetics, Morgan received the Nobel Prize in Physiology or Medicine in 1933.

In the late 1920s, Barbara McClintock (1902–1992) developed chromosomal staining techniques to visualize and differentiate between the different chromosomes of maize (corn). In the 1940s and 1950s, she identified a breakage event on chromosome 9, which she named the dissociation locus (Ds). Ds could change position within the chromosome. She also identified an activator locus (Ac). Ds chromosome breakage could be activated by an Ac element (transposase enzyme). At first, McClintock’s finding of these jumping genes, which we now call transposons, was not accepted by the scientific community. It wasn’t until the 1960s and later that transposons were discovered in bacteriophages, bacteria, and Drosophila. Today, we know that transposons are mobile segments of DNA that can move within the genome of an organism. They can regulate gene expression, protein expression, and virulence (ability to cause disease).

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Microbes and Viruses in Genetic Research

Microbiologists have also played a crucial part in our understanding of genetics. Experimental organisms such as Mendel’s garden peas, Morgan’s fruit flies, and McClintock’s corn had already been used successfully to pave the way for an understanding of genetics. However, microbes and viruses were (and still are) excellent model systems for the study of genetics because, unlike peas, fruit flies, and corn, they are propagated more easily in the laboratory, growing to high population densities in a small amount of space and in a short time. In addition, because of their structural simplicity, microbes and viruses are more readily manipulated genetically.

Fortunately, despite significant differences in size, structure, reproduction strategies, and other biological characteristics, there is biochemical unity among all organisms; they have in common the same underlying molecules responsible for heredity and the use of genetic material to give cells their varying characteristics. In the words of French scientist Jacques Monod, “What is true for E. coli is also true for the elephant,” meaning that the biochemistry of life has been maintained throughout evolution and is shared in all forms of life, from simple unicellular organisms to large, complex organisms. This biochemical continuity makes microbes excellent models to use for genetic studies.

In a clever set of experiments in the 1930s and 1940s, German scientist Joachim Hämmerling (1901–1980), using the single-celled alga Acetabularia as a microbial model, established that the genetic information in a eukaryotic cell is housed within the nucleus. Acetabularia spp. are unusually large algal cells that grow asymmetrically, forming a “foot” containing the nucleus, which is used for substrate attachment; a stalk; and an umbrella-like cap—structures that can all be easily seen with the naked eye. In an early set of experiments, Hämmerling removed either the cap or the foot of the cells and observed whether new caps or feet were regenerated (Figure \(\PageIndex{2}\)). He found that when the foot of these cells was removed, new feet did not grow; however, when caps were removed from the cells, new caps were regenerated. This suggested that the hereditary information was located in the nucleus-containing foot of each cell.

Figure \(\PageIndex{2}\): (a) The cells of the single-celled alga Acetabularia measure 2–6 cm and have a cell morphology that can be observed with the naked eye. Each cell has a cap, a stalk, and a foot, which contains the nucleus. (b) Hämmerling found that if he removed the cap, a new cap would regenerate; but if he removed the foot, a new foot would not regenerate. He concluded that the genetic information needed for regeneration was found in the nucleus. (credit a: modification of work by James St. John)

In another set of experiments, Hämmerling used two species of Acetabularia that have different cap morphologies, A. crenulata and A. mediterranea (Figure \(\PageIndex{3}\)). He cut the caps from both types of cells and then grafted the stalk from an A. crenulata onto an A. mediterranea foot, and vice versa. Over time, he observed that the grafted cell with the A. crenulata foot and A. mediterranea stalk developed a cap with the A. crenulata morphology. Conversely, the grafted cell with the A. mediterranea foot and A. crenulata stalk developed a cap with the A. mediterranea morphology. He microscopically confirmed the presence of nuclei in the feet of these cells and attributed the development of these cap morphologies to the nucleus of each grafted cell. Thus, he showed experimentally that the nucleus was the location of genetic material that dictated a cell’s properties.

Figure \(\PageIndex{3}\): In a second set of experiments, Hämmerling used two morphologically different species and grafted stalks from each species to the feet of the other. He found that the properties of the regenerated caps were dictated by the species of the nucleus-containing foot.

Another microbial model, the red bread mold Neurospora crassa, was used by George Beadle and Edward Tatum to demonstrate the relationship between genes and the proteins they encode. Beadle had worked with fruit flies in Morgan’s laboratory but found them too complex to perform certain types of experiments. N. crassa, on the other hand, is a simpler organism and has the ability to grow on a minimal medium because it contains enzymatic pathways that allow it to use the medium to produce its own vitamins and amino acids.

Beadle and Tatum irradiated the mold with X-rays to induce changes to a sequence of nucleic acids, called mutations. They mated the irradiated mold spores and attempted to grow them on both a complete medium and a minimal medium. They looked for mutants that grew on a complete medium, supplemented with vitamins and amino acids, but did not grow on the minimal medium lacking these supplements. Such molds theoretically contained mutations in the genes that encoded biosynthetic pathways. Upon finding such mutants, they systematically tested each to determine which vitamin or amino acid it was unable to produce (Figure \(\PageIndex{4}\)) and published this work in 1941.

Figure \(\PageIndex{4}\): Beadle and Tatum’s experiment involved the mating of irradiated and nonirradiated mold spores. These spores were grown on both complete medium and a minimal medium to determine which amino acid or vitamin the mutant was unable to produce on its own.

Subsequent work by Beadle, Tatum, and colleagues showed that they could isolate different classes of mutants that required a particular supplement, like the amino acid arginine (Figure \(\PageIndex{5}\)). With some knowledge of the arginine biosynthesis pathway, they identified three classes of arginine mutants by supplementing the minimal medium with intermediates (citrulline or ornithine) in the pathway. The three mutants differed in their abilities to grow in each of the media, which led the group of scientists to propose, in 1945, that each type of mutant had a defect in a different gene in the arginine biosynthesis pathway. This led to the so-called one gene–one enzyme hypothesis, which suggested that each gene encodes one enzyme.

Subsequent knowledge about the processes of transcription and translation led scientists to revise this to the “one gene–one polypeptide” hypothesis. Although there are some genes that do not encode polypeptides (but rather encode for transfer RNAs [tRNAs] or ribosomal RNAs [rRNAs], which we will discuss later), the one gene–one enzyme hypothesis is true in many cases, especially in microbes. Beadle and Tatum’s discovery of the link between genes and corresponding characteristics earned them the 1958 Nobel Prize in Physiology and Medicine and has since become the basis for modern molecular genetics.

Figure \(\PageIndex{5}\): Three classes of arginine mutants were identified, each differing in their ability to grow in the presence of intermediates in the arginine biosynthesis pathway. From this, Beadle and Tatum concluded that each mutant was defective in a different gene encoding a different enzyme in the arginine biosynthesis pathway, leading to them to their one gene–one enzyme hypothesis.

Link to Learning

To learn more about the experiments of Beadle and Tatum, visit this website from the DNA Learning Center.

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Interactive Element

Case Study Preview: “Tacos, Toilets, and Toxins”

Alex thought his spring break trip to Mexico would be unforgettable - and it was, just not in the way he hoped. Two days after returning home, he was stuck on the toilet with cramping and relentless watery diarrhea. The culprit? ETEC, or enterotoxigenic E. coli, better known as the bacterial prankster behind “traveler’s diarrhea.”

In this case, you’ll follow Alex’s misadventure through diagnosis via DNA-based detection of virulence factors, explore how E. coli evolves by swapping genes with other pathogens, and compare ETEC with its much scarier cousin EHEC. You’ll also learn which foods to avoid abroad and why bismuth and bottled water might just save your vacation.

Spoiler alert: it wasn’t the tacos, it was the toxins.

Chapter 10 Case Study - Tacos, Toilets, and Toxins

Key Concepts and Summary

• DNA was discovered and characterized long before its role in heredity was understood. Microbiologists played significant roles in demonstrating that DNA is the hereditary information found within cells.
• In the 1850s and 1860s, Gregor Mendel experimented with true-breeding garden peas to demonstrate the heritability of specific observable traits.
• In 1869, Friedrich Miescher isolated and purified a compound rich in phosphorus from the nuclei of white blood cells; he named the compound nuclein. Miescher’s student Richard Altmann discovered its acidic nature, renaming it nucleic acid. Albrecht Kossell characterized the nucleotide bases found within nucleic acids.
• Although Walter Sutton and Theodor Boveri proposed the Chromosomal Theory of Inheritance in 1902, it was not scientifically demonstrated until the 1915 publication of the work of Thomas Hunt Morgan and his colleagues.
• Using Acetabularia, a large algal cell, as his model system, Joachim Hämmerling demonstrated in the 1930s and 1940s that the nucleus was the location of hereditary information in these cells.
• In the 1940s, George Beadle and Edward Tatum used the mold Neurospora crassa to show that each protein’s production was under the control of a single gene, demonstrating the “one gene–one enzyme” hypothesis.

Footnotes

1. 1 J.G. Mendel. “Versuche über Pflanzenhybriden.” Verhandlungen des naturforschenden Vereines in Brünn, Bd. Abhandlungen 4 (1865):3–7. (For English translation, see http://www.mendelweb.org/Mendel.plain.html)
2. 2 G.W. Beadle, E.L. Tatum. “Genetic Control of Biochemical Reactions in Neurospora.” Proceedings of the National Academy of Sciences 27 no. 11 (1941):499–506.
3. 3 F. Griffith. “The Significance of Pneumococcal Types.” Journal of Hygiene 27 no. 2 (1928):8–159.
4. 4 A.D. Hershey, M. Chase. “Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage.” Journal of General Physiology 36 no. 1 (1952):39–56.