MCB3020 Textbook Chapter 1 PDF

1 The Evolution of Microorganisms and Microbiology ©alex-mit/iStock/Getty Images The Microbial Universe I f you have ever gazed at the night sky on a cloudless evening in a region far from light pollution, you have probably been amazed and perhaps a little humbled by the vast number of stars. It’...

1 The Evolution of Microorganisms and Microbiology ©alex-mit/iStock/Getty Images The Microbial Universe I f you have ever gazed at the night sky on a cloudless evening in a region far from light pollution, you have probably been amazed and perhaps a little humbled by the vast number of stars. It’s hard to estimate just how many stars are out there; best estimates start with our own Milky Way Galaxy, which has roughly 100 billion (1 x 1011) stars. Astronomers figure there is something like 10 trillion (1 x 1013) galaxies in the universe. Assuming all galaxies are roughly the size of the Milky Way, you wind up with a number around 1 x 1024 stars in the universe—not the most accurate number, but nonetheless daunting. If you turn your gaze back to Earth, however, you will find even more awesome abundance. For example, if you stacked all of the 1 x 10 31 viruses on Earth one on top of the other, they would stretch about 100 million light-years. That’s about 43 times further away than the Andromeda Galaxy. And next time you take a dip in the sea, consider that there are at least a hundred thousand (1 x 105) more bacteria in the ocean (about 1.3 x 1029) than stars in the universe. Or perhaps you prefer staying on land where a teaspoon of soil has about a billion (1 x 10 9) microorganisms. What are all these microorganisms doing? The short answer is, making life for the rest of us possible. How? Starting about 2.4 billion years ago, bacteria called cyanobacteria were the first organisms to release oxygen in abundance into the atmosphere. This has been dubbed the “great oxidation event,” and it set the stage for oxygen-consuming organisms (like us) to evolve. Microorganisms have another starring role in the evolution of life because only bacteria can fix nitrogen—that is, take gaseous nitrogen and convert it to organic nitrogen used by plants, animals, and other microbes. Finally, can you imagine what life on Earth would look like if dead organic material were not degraded? Probably best not to. Much better to think of foods like beer, wine, chocolate, cheese, and yogurt—all microbial products. But of course not all microorganisms make life possible, or even easier. Each year about 16 million (1.6 x 107 ) people die from infectious disease; and many of these deaths are preventable by either vaccination or antibiotic treatments. Ironically most vaccines and antibiotics are also microbial products. Although we know the most about disease-causing microorganisms, because less than 1% of all microorganisms cause disease, there is a lot left to learn about microbes. In fact, like the number of stars, the number of microbial species (including bacteria, viruses, fungi, and protists) is actively debated. What is certain is that microbes are important for the life of the planet and its plants and animals. Our goal in this chapter is to introduce you to this amazing world of microorganisms and to outline the history of their evolution and discovery. Microbiology is a biological science, and as such, much of what you will learn in this text is similar to what you have learned in high school and college biology classes that focus on large organisms. But microbes have unique properties, so microbiology has unique approaches to understanding them. These too will be introduced. But before you delve into this chapter, check to see if you have the background needed to get the most from it. Readiness Check: Based on what you have learned previously, you should be able to: ✓ List the features of eukaryotic cells that distinguish them from other cell types ✓ Understand the basic structure of the macromolecules, nucleic acids, proteins, carbohydrates, and lipids (see appendix I) 1.1 Members of the Microbial World After reading this section, you should be able to: a. Define the term microbiology b. Explain Carl Woese’s contributions in establishing the three-domain system for classifying cellular life c. Determine the type of microbe (bacterium, fungus, etc.) when given a description of a newly discovered one d. Provide an example of the importance to humans of each of the major types of microbes Microorganisms are defined as those organisms too small to be seen clearly by the unaided eye (figure 1.1). They are generally 1 millimeter or less in diameter. Although small size is an important characteristic of microbes, it is not sufficient to define them. 1 wil11886_ch01_001-019.indd 1 23/10/18 9:23 am 2 CHAPTER 1 | The Evolution of Microorganisms and Microbiology Organisms and biological entities studied by microbiologists can be Cellular Acellular includes includes Fungi Protists Bacteria Archaea Viruses Viroids Satellites Prions e.g. e.g. e.g. e.g. composed of composed of composed of composed of Yeasts Molds Algae Protozoa Slime molds Escherichia coli Methanogens Protein and nucleic acid RNA Nucleic acid enclosed in a protein shell Protein Figure 1.1 Concept Map Showing the Types of Biological Entities Studied by Microbiologists. MICRO INQUIRY How would you alter this concept map so that it also distinguishes cellular organisms from each other? Some microbes, such as bread molds and filamentous photosynthetic microbes (e.g., “pond scum”), are actually visible without microscopes. These macroscopic microbes often consist of small aggregations of cells. Some macroscopic microorganisms are multicellular. They are distinguished from other multicellular life forms such as plants and animals by their lack of highly differentiated tissues. Most unicellular microbes are microscopic. In summary, cellular microbes are usually smaller than 1 millimeter in diameter, often unicellular and, if multicellular, lack differentiated tissues. In addition to microorganisms, microbiologists study a variety of acellular biological entities (figure 1.1). These include viruses and subviral agents. The terms “microorganism” and “microbe” are sometimes applied to these acellular agents as well. The diversity of microorganisms has always presented a challenge to microbial taxonomists. Early descriptions of cellular microbes as either plants or animals were too simple. For instance, some microbes are motile like animals but also have cell walls and are photosynthetic like plants. An important breakthrough in microbial taxonomy arose from studies of their cellular architecture, when it was discovered that cells exhibited one of two possible “floor plans.” Cells that came to be called prokaryotic cells (Greek pro, before; karyon, nut or kernel) have an open floor plan. That is, their contents are not divided into compartments (“rooms”) by membranes. Only eukaryotic cells (Greek eu, true) have a nucleus and other membrane-bound organelles (e.g., mitochondria, chloroplasts) that separate some cellular materials and processes from others. wil11886_ch01_001-019.indd 2 These observations eventually led to the development of a classification scheme that divided organisms into five kingdoms: Monera, Protista, Fungi, Animalia, and Plantae. Microorganisms (except for viruses and other acellular infectious agents, which have their own classification system) were placed in the first three kingdoms. In this scheme, all organisms with prokaryotic cell structure were placed in Monera. The five-kingdom system was an important development in microbial taxonomy, but it is no longer accepted by microbiologists. This is because “prokaryotes” are too diverse to be grouped together in a single kingdom. Furthermore, it is currently argued that the term prokaryote is not meaningful and should be abandoned. Use of the term “prokaryote” is controversial (section 3.1) Great progress has been made in three areas that profoundly affect microbial classification. First, much has been learned about the detailed structure of microbial cells from the use of electron microscopy. Second, microbiologists have determined the biochemical and physiological characteristics of many different microorganisms. Third, the sequences of nucleic acids and proteins from a wide variety of organisms have been compared. The comparison of ribosomal RNA (rRNA) nucleic acid sequences, begun by Carl Woese (1928–2012) in the 1970s, was instrumental in demonstrating that there are two very different groups of organisms with prokaryotic cell architecture: Bacteria and Archaea. Later studies based on rRNA comparisons showed that Protista is not a cohesive taxonomic unit (i.e., taxon) and that it should be divided into three or more kingdoms. These studies and others led many taxonomists to reject the fivekingdom system in favor of one that divides cellular organisms 23/10/18 9:23 am 1.1 Members of the Microbial World 3 Ve mm rruc ati om m ic Ba onad robi cte roid etes a ete s Chlo rofle xi og ot m ri a ond och als im Fu ng i mit An Ge Cy ch anob lo a ro cter Pr ia pl ot as eo ts ba ct er ia acteria i Acidob b oro s Chl e icut Firm ae er Th Actinobacteria Bacteria Origin Crenarchaeota Archaea Euryarchaeota s te a ell g fla He no e oa lga Ch d A Re ts Plan Stramenopiles Alveolates Acanth amoe bae Eu gle no zoa Trich omo nads Diplom onads a se Eukarya o ob ol Unresolved branching order r te rRNA sequence change Figure 1.2 Universal Phylogenetic Tree. These evolutionary relationships are based on rRNA sequence comparisons. Only representative lineages have been identified. MICRO INQUIRY How many of the taxa listed in the figure include microbes? into three domains: Bacteria, Archaea, and Eukarya (all eukaryotic organisms) (figure 1.2). Nucleic acids (appendix I); Proteins (appendix I) Members of domain Bacteria are usually single-celled organisms.1 Most have cell walls that contain the structural molecule peptidoglycan. Bacteria are abundant in soil, water, and air, including sites that have extreme temperatures, pH, or salinity. Bacteria are also major inhabitants of our bodies, forming the human microbiome. Indeed, more microbial cells are found in and on the human body than there are human cells. These microbes begin to colonize humans shortly after birth. As the microbes establish themselves, they contribute to the development of the body’s immune system. Those microbes that inhabit the large intestine help the body digest food and produce vitamins. In these and many other ways, the human microbiome helps maintain our health and well-being. Overview of bacterial cell wall structure (section 3.4); The microbe-human ecosystem (chapter 34) Unfortunately some bacteria cause disease, and some of these diseases have had a huge impact on human history. In 1347 the plague (Black Death), a disease carried by bacteria living in fleas, struck Europe with brutal force, killing one-third of the population within 4 years. Over the next 80 years, the disease struck repeatedly, eventually wiping out roughly half of the European population. The plague’s effect was so great that most historians believe it changed European culture and prepared the way for the Renaissance. Members of domain Archaea are distinguished from bacteria by many features, most notably their distinctive rRNA sequences, lack of peptidoglycan in their cell walls, and unique membrane lipids. Some have unusual metabolic characteristics, such as the ability to generate methane (natural) gas. Many archaea are found in extreme environments, including those with high temperatures (thermophiles) and high concentrations of salt (extreme halophiles). Although some archaea are members of a community of microbes involved in gum disease in humans, their role in causing disease has not been clearly established. Domain Eukarya includes plants, animals, and microorganisms classified as protists or fungi. Protists are generally unicellular but larger than most bacteria and archaea. They have traditionally been divided into protozoa and algae. However, these terms lack taxonomic value because protists, algae, and protozoa do not form three groups, each with a single evolutionary history. Nonetheless, for convenience, we use these terms here. The major types of protists are algae, protozoa, slime molds, and water molds. Algae are photosynthetic. They, together with cyanobacteria, produce about 50% of the planet’s oxygen and are the foundation of aquatic food chains. Protozoa are usually motile and many free-living protozoa function as the principal hunters and grazers of the microbial world. They obtain nutrients by ingesting organic matter and other microbes. They can be found in many different environments, and some are normal inhabitants of the intestinal tracts of animals, where they aid in digestion of complex materials such as cellulose. A few cause disease in humans and other animals. Slime molds are protists that behave like protozoa in one stage of their life cycle but like fungi in another. In the protozoan phase, they hunt for and engulf food particles, consuming decaying vegetation and other microbes. Water molds are protists that grow on the surface of freshwater and moist soil. They feed on decaying plant material. Some water molds have produced devastating plant infections, including the Great Potato Famine of 1846–1847 in Ireland, which led to the mass exodus of Irish to the United States and other countries. Although slime and water molds are protists, they were once thought to be fungi, thus the confusing nomenclature. Protists (chapter 24) Fungi are a diverse group of microorganisms that range from unicellular forms (yeasts) to molds and mushrooms. Molds and mushrooms are multicellular fungi that form thin, threadlike structures called hyphae. They absorb all their nutrients from their environment. Because of their metabolic capabilities, many fungi play beneficial roles, including making bread dough rise, producing antibiotics, and decomposing dead organisms. Some fungi associate with plant roots to form mycorrhizae. Mycorrhizal fungi transfer nutrients to the roots, improving growth of the plants, especially in poor soils. Other fungi cause plant diseases (e.g., rusts, powdery mildews, and smuts) and diseases in humans and other animals. Fungi (chapter 25) 1 In this text, the term bacteria (s., bacterium) is used to refer to those microbes belonging to domain Bacteria, and the term archaea (s., archaeon) is used to refer to those that belong to domain Archaea. In some publications, the term bacteria is used to refer to all cells having prokaryotic cell structure. That is not the case in this text. wil11886_ch01_001-019.indd 3 23/10/18 9:23 am 4 CHAPTER 1 | The Evolution of Microorganisms and Microbiology The microbial world also includes numerous acellular infectious agents. Viruses are acellular entities that must invade a host cell to multiply. The simplest virus particles (also called virions) are composed only of proteins and a nucleic acid, and can be extremely small (the smallest is 10,000 times smaller than a typical bacterium). However, their small size belies their power. They cause many animal and plant diseases and have caused epidemics that have shaped human history. Viral diseases include smallpox, rabies, influenza, AIDS, the common cold, and some cancers. Viruses also play important roles in aquatic environments, where they play a critical role in shaping aquatic microbial communities. Viroids are infectious agents composed only of ribonucleic acid (RNA). They cause numerous plant diseases. Satellites are composed of a nucleic acid enclosed in a protein shell. They must coinfect a host cell with virus, called a helper virus, to complete their life cycle. Viroids, on the other hand, cause only plant diseases while satellites and their helper viruses cause both plant and animal diseases. Finally, prions, infectious agents composed only of protein, are responsible for causing a variety of spongiform encephalopathies such as scrapie and “mad cow disease.” Viruses and other acellular infectious agents (chapter 6) Comprehension Check 1. How did the methods used to classify microbes change, particularly in the last half of the twentieth century? What was the result of these technological advances? 2. Identify one characteristic for each of these types of microbes that distinguishes it from the other types: bacteria, archaea, protists, fungi, viruses, viroids, satellites, and prions. 1.2 Microbes Have Evolved and Diversified for Billions of Years After reading this section, you should be able to: a. Propose a timeline of the origin and history of microbial life and integrate supporting evidence into it b. Design a set of experiments that could be used to place a newly discovered cellular microbe on a phylogenetic tree based on small subunit (SSU) rRNA sequences c. Compare and contrast the definitions of plant and animal species, microbial species, and microbial strains A review of figure 1.2 reminds us that in terms of the number of taxa, microbes are the dominant organisms on Earth. How has microbial life been able to radiate to such an astonishing level of diversity? To answer this question, we must consider microbial evolution. The field of microbial evolution, like any other scientific endeavor, is based on the formulation of hypotheses, the gathering and analysis of data, and the reformation of hypotheses wil11886_ch01_001-019.indd 4 based on newly acquired evidence. That is to say, the study of microbial evolution is based on the scientific method. To be sure, it is difficult to amass evidence when considering events that occurred millions, and often billions, of years ago, but the application of molecular methods has revealed a living record of life’s ancient history. This section describes the outcome of this scientific research. Theories of the Origin of Life Depend Primarily on Indirect Evidence Dating meteorites through the use of radioisotopes places our planet at an estimated 4.5 to 4.6 billion years old. However, conditions on Earth for the first 100 million years or so were far too harsh to sustain any type of life. Eventually bombardment by meteorites decreased, water appeared on the planet in liquid form, and gases were released by geological activity to form Earth’s atmosphere. These conditions were amenable to the origin of the first life forms. But how did this occur, and what did these life forms look like? To find evidence of life and to develop hypotheses about its origin and subsequent evolution, scientists must be able to define life. Although even very young children can examine an object and correctly determine whether it is living or not, defining life succinctly has proven elusive for scientists. Thus most definitions of life consist of a set of attributes. The attributes of particular importance to paleobiologists are an orderly structure, the ability to obtain and use energy (i.e., metabolism), and the ability to reproduce. Just as NASA scientists are using the characteristics of microbes on Earth today to search for life elsewhere, so too are scientists examining extant organisms, those organisms present today, to explore the origin of life. Some extant organisms have structures and molecules that represent “relics” of ancient life forms. These can provide scientists with ideas about the type of evidence to seek when testing hypotheses. The best direct evidence for the nature of primitive life would be a fossil record. There have been reports of microbial fossil discoveries since 1977 (figure 1.3). These have always met with skepticism because finding them involves preparing thin slices of ancient rocks and examining the slices for objects that look like cells. Unfortunately some things that look like cells can be formed by geological forces that occurred as the rock was formed. The result is that the fossil record for microbes is sparse and always open to reinterpretation. Despite these problems most scientists agree that life was present on Earth about 3.5–3.7 billion years ago (figure 1.4). To reach this conclusion, biologists rely primarily on indirect evidence. Among the indirect evidence used are molecular fossils. These are chemicals found in rock or sediment that are chemically related to molecules found in cells. For instance, the presence in a rock of molecules called hopanes is an indication that when the rock was formed, bacteria were present. This conclusion is reached because hopanes are formed from hopanoids, which are found in the plasma membranes of extant bacteria. As you can see, no single piece of evidence can 23/10/18 9:23 am 1.2 Microbes Have Evolved and Diversified for Billions of Years 5 stand alone. Instead many pieces of evidence are put together in an attempt to get a coherent picture to emerge, much as for a jigsaw puzzle. Early Life Was Probably RNA Based The origin of life rests on a single question: How did early cells arise? At a minimum, modern cells consist of a plasma membrane enclosing water in which numerous chemicals are dissolved and subcellular structures float. It seems likely that the first self-replicating entity was much simpler than even the most primitive modern living cells. Before there was life, most evidence suggests that Earth was a Figure 1.3 Possible Microfossils Found in the Archaeon Apex Chert of Australia. Chert is a type of granular sedimentary rock rich in silica. These structures were discovered in 1977. Because of their similarity to filamentous cyanobacteria they were proposed to be microfossils. In 2011 scientists reported that similar structures from the same chert were not biological in origin. They used spectrometry and microscopy techniques not available in 1977 to show that the structures were fractures in the rock filled with quartz and hematite. Scientists are still debating whether or not these truly are microfossils. ©J. William Schopf very different place: hot and anoxic, with an atmosphere rich in water vapor, carbon dioxide, and nitrogen. In the oceans, hydrogen, methane, and carboxylic acids were formed by geological and chemical processes. Areas near hydrothermal vents or in shallow pools may have provided the conditions that allowed chemicals to react with one another, randomly “testing” the usefulness of the reaction and the stability of its products. Some reactions released energy and would eventually become the basis of modern cellular metabolism. Other reactions generated molecules that functioned as catalysts, some aggregated with other molecules to form the predecessors of modern cell structures, and others were able to replicate and act as units of hereditary information. In modern cells, three different molecules fulfill the roles of catalysts, structural molecules, and hereditary molecules (figure 1.5). Proteins have two major roles in modern cells: catalytic and structural. Catalytic proteins are enzymes wil11886_ch01_001-019.indd 5 and structural proteins serve a myriad of functions such as transport, attachment, and motility. DNA stores hereditary information that is replicated and passed on to the next generation. RNA is involved in converting the information stored in DNA into protein. Any hypothesis about the origin of life must account for the evolution of these molecules, but the very nature of their relationships to each other in modern cells complicates attempts to imagine how they evolved. As demonstrated in figure 1.5, proteins can do cellular work, but their synthesis involves other proteins and RNA, and uses information stored in DNA. DNA can’t do cellular work and proteins are needed for its replication. RNA synthesis requires both DNA as the template and proteins as the catalysts for the reaction. Based on these considerations, it is hypothesized that at some time in the evolution of life, there must have been a single molecule that could do both cellular work and replicate. This idea was supported in 1981 when Thomas Cech discovered a catalytic RNA molecule in a protist (Tetrahymena sp.) that could cut out an internal section of itself and splice the remaining sections back together. Since then, other catalytic RNA molecules have been discovered, including an RNA found in ribosomes that is responsible for forming peptide bonds—the bonds that hold together amino acids, the building blocks of proteins. Catalytic RNA molecules are now called ribozymes. The discovery of ribozymes suggested that RNA at some time was capable of storing, copying, and expressing genetic information, as well as catalyzing other chemical reactions. In 1986 Nobel laureate Walter Gilbert coined the term RNA world to describe this precellular stage in the evolution of life. However, for this precellular RNA-based stage to proceed to the evolution of cellular life forms, a lipid membrane must have formed around the RNA (figure 1.6). This important evolutionary step is easier to imagine than other events in the origin of cellular life forms because lipids, major structural components of the membranes of modern organisms, spontaneously form liposomes—vesicles bounded by a lipid bilayer. Lipids (appendix I) Jack Szostak, also a Noble laureate, is a leader in exploring how RNA-containing cells, so-called protocells, may have formed. When his group created liposomes using simpler fatty acids than those found in membranes today, the liposomes were leaky. These leaky liposomes allowed single RNA nucleotides to move into the liposome, but prevented large RNA chains from moving out. Furthermore, researchers could prod the liposomes into growing and dividing. Dr. Szostak’s group has also been able to create conditions in which an RNA molecule could serve as a template for synthesis of a complementary RNA strand. Thus their experiments may have recapitulated the early steps of the evolution of cells. As seen in figure 1.6, several other processes would need to occur to reach the level of complexity found in extant cells. Apart from its ability to perform catalytic activities, the function of RNA suggests its ancient origin. Consider that much 23/10/18 9:23 am PHANEROZOIC 144 206 248 290 354 417 443 Periods 65 Eras Eons 0 1.8 CENOZOIC Millions of Years Ago | The Evolution of Microorganisms and Microbiology Quaternary MESOZOIC CHAPTER 1 Cretaceous Tertiary 7 mya—Hominids first appear. Jurassic Triassic 225 mya—Dinosaurs and mammals first appear. Permian PALEOZOIC 6 490 Carboniferous Silurian Ordovician Cambrian LATE 543 300 mya—Reptiles first appear. Devonian 450 mya—Large terrestrial colonization by plants and animals. 520 mya—First vertebrates; first land plants. 533–525 mya—Cambrian explosion creates diverse animal life. 1.5 bya—Multicellular eukaryotic organisms first appear. LATE MIDDLE 3,000 2.5–2.0 bya—Eukaryotic cells with mitochondria or chloroplasts first appear. ARCHAEAN 2,500 PRECAMBRIAN EARLY 1,600 MIDDLE PROTEROZOIC 900 EARLY 3,400 3,800 3.7 bya—Fossils of primitive microbes. HADEAN 3.8–3.5 bya—First cells appear. 4,550 Figure 1.4 An Overview of the History of Life on Earth. mya = million years ago; bya = billion years ago. wil11886_ch01_001-019.indd 6 23/10/18 9:23 am 1.2 Microbes Have Evolved and Diversified for Billions of Years 7 Stores ©Benny Marty/ Shutterstock Serves as template for synthesis of new DNA Encodes sequence of nucleotides in Genetic information Prebiotic soup Liposome RNA Catalyzes synthesis of RNA Regulates expression of Functions in synthesis of Catalyzes synthesis of Probiont: RNA only Encodes sequence of amino acids in Protein Involved in synthesis of more Forms Catalyzes Structures Chemical reactions Probiont: RNA and proteins Figure 1.5 Functions of DNA, RNA, and Protein, and Their Relationships to Each Other in Extant Cells. of the cellular pool of RNA in modern cells exists in the ribosome, a structure that consists largely of rRNA and uses messenger RNA (mRNA) and transfer RNA (tRNA) to construct proteins. Also rRNA itself catalyzes peptide bond formation during protein synthesis. Thus RNA seems to be well poised for its importance in the development of proteins. Because RNA and DNA are structurally similar, RNA could have given rise to double-stranded DNA. It is suggested that once DNA evolved, it became the storage facility for genetic information because it provided a more chemically stable structure. Two other pieces of evidence support the RNA world hypothesis: the fact that the energy currency of cells, ATP, is a ribonucleotide and the discovery that RNA can regulate gene expression. So it would seem that proteins, DNA, and cellular energy can be traced back to RNA. ATP: the major energy currency of cells (section 10.2); Riboswitches (section 14.3); Translational riboswitches (section 14.4) Despite evidence supporting the RNA world hypothesis, it is not without problems, and many argue against it. wil11886_ch01_001-019.indd 7 Cellular life: RNA, DNA, and proteins Figure 1.6 The RNA World Hypothesis for the Origin of Life. MICRO INQUIRY Why are the probionts pictured above not considered cellular life? Another area of research also fraught with considerable debate is the evolution of metabolism, in particular the evolution of energy-conserving metabolic processes. The early Earth was a hot environment that lacked oxygen. Thus the cells that arose there must have been able to use the available energy sources under these harsh conditions. Today there are heatloving archaea capable of using inorganic molecules such as FeS as a source of energy. Some suggest that this interesting metabolic capability is a remnant of the first form of energy metabolism. Another metabolic strategy, oxygen-releasing photosynthesis (oxygenic photosynthesis), appears to have evolved perhaps as early as 2.7 billion years ago. Fossils of cyanobacteria-like cells found in rocks dating to that time 23/10/18 9:23 am 8 CHAPTER 1 | The Evolution of Microorganisms and Microbiology (a) (b) Figure 1.7 Stromatolites. (a) Section of a fossilized stromatolite. Evolutionary biologists think the layers of material were formed when mats of cyanobacteria, layered one on top of the other, became mineralized. (b) Modern stromatolites from Western Australia. Each stromatolite is a rocklike structure, typically 1 m in diameter, containing layers of cyanobacteria. (a) ©Dirk Wiersma/SPL/Science Source; (b) ©Horst Mahr/age fotostock support this hypothesis, as does the discovery of ancient stromatolites (figure 1.7a). Stromatolites are layered rocks formed by the incorporation of mineral sediments into layers of microorganisms growing in thick mats on surfaces (figure 1.7b). The appearance of cyanobacteria-like cells was an important step in the evolution of life on Earth. The oxygen they released is thought to have altered Earth’s atmosphere to its current oxygen-rich state, allowing the evolution of additional energy-capturing strategies such as aerobic respiration, the oxygen-consuming metabolic process that is used by many microbes and animals. Evolution of the Three Domains of Life As noted in section 1.1, rRNA comparisons were an important breakthrough in the classification of microbes; this analysis also provides insights into the evolutionary history of all life. What began with the examination of rRNA from relatively few organisms has been expanded by the work of many others, including Norman Pace. Dr. Pace has developed a universal phylogenetic tree (figure 1.2) based on comparisons of small subunit rRNA molecules (SSU rRNA), the rRNA found in the small subunit of the ribosome. Here we examine how these comparisons are made and what the universal phylogenetic tree tells us. Bacterial ribosomes (section 3.6); Microbial taxonomy and phylogeny are largely based on molecular characterization (section 19.3) Comparing SSU rRNA Molecules The details of phylogenetic tree construction are discussed in chapter 19. However, the general concept is not difficult to understand. In one approach, the sequences of nucleotides in the genes that encode SSU rRNAs from diverse organisms are aligned, and pair-wise comparisons of the sequences are wil11886_ch01_001-019.indd 8 made. For each pair of SSU rRNA gene sequences, the number of differences in the nucleotide sequences is counted (figure 1.8). This value serves as a measure of the evolutionary distance between the organisms; the more differences counted, the greater the evolutionary distance. The evolutionary distances from many comparisons are used by sophisticated computer programs to construct the tree. The tip of each branch in the tree represents one of the organisms used in the comparison. The distance from the tip of one branch to the tip of another is the evolutionary distance between the two organisms. Two things should be kept in mind when examining phylogenetic trees developed in this way. The first is that they are molecular trees, not organismal trees. In other words, they represent, as accurately as possible, the evolutionary history of a molecule and the gene that encodes it. Second, the distance between branch tips is a measure of relatedness, not of time. If the distance along the lines is very long, then the two organisms are more evolutionarily diverged (i.e., less related). However, we do not know when they diverged from each other. This concept is analogous to a printed map that accurately shows the distance between two cities but because of many factors (traffic, road conditions, etc.) cannot show the time needed to travel that distance. LUCA What does the universal phylogenetic tree tell us about the evolution of life? At the center of the tree is a line labeled “Origin” (figure 1.2). This is where data indicate the last universal common ancestor (LUCA) to all three domains should be placed. LUCA is on the bacterial branch, which means that Archaea and Eukarya evolved independently, separate from Bacteria. Thus the universal phylogenetic tree presents a picture in which all 23/10/18 9:23 am 1.2 Microbes Have Evolved and Diversified for Billions of Years 9 Cells from organism 1 Lyse cells to release contents and isolate DNA. DNA Use polymerase chain reaction to amplify and purify SSU rRNA genes. SSU rRNA genes Sequence genes. ATGCTCAAGTCA Repeat process for other organisms. Align sequences to be compared. SSU rRNA sequence Organism ATGCTCAAGTCA TAGCTCGTGTAA AAGCTCTAGTTA AACCTCATGTTA 1 2 3 4 Mitochondria, Mitochondria-Like Organelles, and Chloroplasts Evolved from Endosymbionts Count the number of nucleotide differences between each pair of sequences and calculate evolutionary distance (ED). Pair compared 1 2 1 3 Corrected ED ED 0.42 0.61 0.25 0.30 1 2 2 3 0.33 0.33 0.33 0.25 4 3 4 4 0.44 0.44 0.44 0.30 For organisms 1 and 2, 5 of the 12 nucleotides are different: ED = 5/12 = 0.42. The initial ED calculated is corrected using a statistical method that considers for each site the probability of a mutation back to the original nucleotide or of additional forward mutations. Computer analysis is used to construct phylogenetic tree. 3 0.08 1 0.08 Unrooted phylogenetic tree. Note that distance from one tip to another is proportional to the ED. 0.23 2 0.15 0.30 4 Figure 1.8 The Construction of Phylogenetic Trees Using a Distance Method. The polymerase chain reaction is described in chapter 17. MICRO INQUIRY Why does the branch length indicate amount of evolutionary change but not the time it took for that change to occur? wil11886_ch01_001-019.indd 9 life, regardless of eventual domain, arose from a single common ancestor. One can envision the universal tree of life as a real tree that grows from a single seed. The evolutionary relationship of Archaea and Eukarya is still the matter of considerable debate. According to the universal phylogenetic tree we show here, Archaea and Eukarya shared common ancestry but diverged and became separate domains. Other versions suggest that Eukarya evolved out of Archaea. The close evolutionary relationship of these two forms of life is still evident in the manner in which they process genetic information. For instance, certain protein subunits of archaeal and eukaryotic RNA polymerases, the enzymes that catalyze RNA synthesis, resemble each other to the exclusion of those of bacteria. However, archaea have other features that are most similar to their counterparts in bacteria (e.g., mechanisms for conserving energy). This has further complicated and fueled the debate. The evolution of the nucleus and endoplasmic reticulum is also at the center of many controversies. However, hypotheses regarding the evolution of other membrane-bound organelles are more widely accepted and are considered next. The endosymbiotic hypothesis is generally accepted as the origin of several eukaryotic organelles, including mitochondria, chloroplasts, and hydrogenosomes. Endosymbiosis is an interaction between two organisms in which one organism lives inside the other. The original endosymbiotic hypothesis proposed that over time a bacterial endosymbiont of an ancestral cell in the eukaryotic lineage lost its ability to live independently, becoming either a mitochondrion, if the intracellular bacterium used aerobic respiration, or a chloroplast, if the endosymbiont was a photosynthetic bacterium (see figure 19.7). Although the mechanism by which the endosymbiotic relationship was established is unknown, there is considerable evidence to support the hypothesis. Mitochondria and chloroplasts contain DNA and ribosomes; both are similar to bacterial DNA and ribosomes. Peptidoglycan, the unique bacterial cell wall molecule, has even been found between the two membranes that enclose the chloroplasts of some algae. Indeed, inspection of figure 1.2 shows that both organelles belong to the bacterial lineage based on SSU rRNA analysis. More specifically, mitochondria are most closely related to bacteria called proteobacteria. The chloroplasts of plants and green algae are thought to have descended from an ancestor of the cyanobacterial genus Prochloron, which contains species that live within marine invertebrates. Phylum Cyanobacteria: oxygenic photosynthetic bacteria (section 21.4); The proteobacterial origin of mitochondria (section 22.1) Recently the endosymbiotic hypothesis for mitochondria has been modified by the hydrogen hypothesis. This asserts that the endosymbiont was an anaerobic bacterium that produced H2 and CO2 as end products of its metabolism. Over time, 23/10/18 9:23 am 10 CHAPTER 1 | The Evolution of Microorganisms and Microbiology the host became dependent on the H2 produced by the endosymbiont. Ultimately the endosymbiont evolved into one of several organelles (see figure 5.13). If the endosymbiont developed the capacity to perform aerobic respiration, it evolved into a mitochondrion. Other endosymbionts evolved into other organelles such as a hydrogenosome—an organelle that produces ATP by a process called fermentation found in some extant protists (see figure 5.15). Evolution of Cellular Microbes Although the history of early cellular life forms may never be known, we know that once they arose, they were subjected to the same evolutionary processes as modern organisms. The ancestral bacteria, archaea, and eukaryotes possessed genetic information that could be duplicated, lost, or mutated in other ways. These mutations could have many outcomes. Some led to the death of the mutant microbe, but others allowed new functions and characteristics to evolve. Those mutations that allowed the organism to increase its reproductive ability were selected and passed on to subsequent generations. In addition to selective forces, geographic isolation of populations allowed some groups to evolve separately from others. Thus selection and isolation led to the eventual development of new collections of genes (i.e., genotypes) and many new species. In addition to mutation, other mechanisms exist for reconfiguring the genotypes of a species and therefore creating genetic diversity. Most eukaryotic species increase their genetic diversity by reproducing sexually. Thus each offspring of the two parents has a mixture of parental genes and a unique genotype. Bacteria and archaea do not reproduce sexually. They increase their genetic diversity by mutation and horizontal (or lateral) gene transfer (HGT). During HGT, genetic information from a donor organism is transferred to a recipient, creating a new genotype. Thus genetic information is passed between individuals of the same generation and even between species found in different domains of life. Genome sequencing has revealed that HGT has played an important role in the evolution of all microbial species. Importantly, HGT still occurs in bacteria and archaea leading to the rapid evolution of microoganisms with antibiotic resistance, new virulence properties, and novel metabolic capabilities. The outcome of HGT is that most microbes have mosaic genomes composed of bits and pieces of the genomes of other organisms. Horizontal gene transfer: creating genetic variation the asexual way (section 16.4) Microbial Species All students of biology are introduced early in their careers to the concept of a species. But the term has different meanings for sexual and asexual organisms. Taxonomists working with plants and animals define a species as a group of interbreeding or potentially interbreeding natural populations that is reproductively isolated from other groups. This definition also is appropriate for the many eukaryotic microbes that wil11886_ch01_001-019.indd 10 reproduce sexually. However, bacterial and archaeal species cannot be defined by this criterion, since they do not reproduce sexually. Therefore, comparisons of genome sequences are often used to distinguish one species from another. An appropriate definition is currently the topic of considerable discussion. A common definition is that bacterial and archaeal species are a collection of strains that share many stable properties and differ significantly from other groups of strains. A strain consists of the descendants of a single, pure microbial culture. Strains within a species may be described in a number of different ways. Biovars are variant strains characterized by biochemical or physiological differences, morphovars differ morphologically, serovars have distinctive properties that can be detected by antibodies, and pathovars are pathogenic strains distinguished by the plants in which they cause disease. What is a microbial species? (section 19.5) Microbiologists name microbes using the binomial system of the eighteenth-century biologist and physician Carl Linnaeus. The Latinized, italicized name consists of two parts. The first part, which is capitalized, is the generic name (i.e., the name of the genus to which the microbe belongs), and the second is the uncapitalized species epithet. For example, the bacterium that causes plague is called Yersinia pestis. Often the name of an organism will be shortened by abbreviating the genus name with a single capital letter (e.g., Y. pestis). Comprehension Check 1. Describe two reasons RNA is thought to be the first self-replicating biomolecule. 2. Explain the endosymbiotic hypothesis of the origin of mitochondria, hydrogenosomes, and chloroplasts. List two pieces of evidence that support this hypothesis. 3. What is the difference between mutation and horizontal gene transfer? 4. What is the correct way to write this microbe’s name: bacillus subtilis, Bacillus subtilis, Bacillus Subtilis, or Bacillus subtilis? Identify the genus name and the species epithet. 1.3 Microbiology Advanced as New Tools for Studying Microbes Were Developed After reading this section, you should be able to: a. Evaluate the importance of the contributions to microbiology made by Hooke, Leeuwenhoek, Pasteur, Lister, Koch, Beijerinck, von Behring, Kitasato, Metchnikoff, and Winogradsky b. Outline a set of experiments that might be used to decide if a particular microbe is the causative agent of a disease c. Predict the difficulties that might arise when using Koch’s postulates to determine if a microbe causes a disease unique to humans 23/10/18 9:23 am 1.3 Microbiology Advanced as New Tools for Studying Microbes Were Developed 11 Even before microorganisms were seen, some investigators suspected their existence and role in disease. Among others, the Roman philosopher Lucretius (about 98–55 BCE) and the physician Girolamo Fracastoro (1478–1553) suggested that disease was caused by invisible living creatures. However, until microbes could actually be seen and studied in some other way, their existence remained a matter of conjecture. Therefore microbiology is defined not only by the organisms it studies but also by the tools used to study them. The development of microscopes was the critical first step in the evolution of the discipline. However, microscopy alone is unable to answer the many questions microbiologists ask about microbes. A distinct feature of microbiology is that microbiologists often remove microorganisms from their normal habitats and grow them isolated from other microbes. This is called a pure or axenic culture. The development of techniques for isolating microbes in pure culture was another critical step in microbiology’s history. However, it is now recognized as having limitations. Microbes in pure culture are in some ways like animals in a zoo; just as a zoologist cannot fully understand the ecology of animals by studying them in zoos, microbiologists cannot fully understand microbes by studying them in pure culture. Today molecular genetic techniques and genomic analyses are providing new insights into the lives of microbes. Methods in microbial ecology (chapter 29); Microbial genomics (chapter 18) Here we describe how the tools used by microbiologists have influenced the development of the field. As microbiology evolved as a science, it contributed greatly to the well-being of humans. This is exemplified by the number of microbiologists who have won the Nobel Prize. The historical context of some of the important discoveries in microbiology is shown in figure 1.9. Microscopy Led to the Discovery of Microorganisms The earliest microscopic observations of organisms appear to have been made between 1625 and 1630 on bees and weevils by the Italian Francesco Stelluti (1577–1652), using a microscope probably supplied by Galileo (1564–1642). Robert Hooke (1635–1703) is credited with publishing the first drawings of microorganisms in the scientific literature. In 1665 he published a highly detailed drawing of the fungus Mucor in his book Micrographia. Micrographia is important not only for its exquisite drawings but also for the information it provided on building microscopes. One design discussed in Micrographia was probably a prototype for the microscopes built and used by the amateur microscopist Antony van Leeuwenhoek (1632–1723) of Delft, the Netherlands (figure 1.10a). Leeuwenhoek earned his living as a draper and haberdasher (a dealer in men’s clothing and accessories) but spent much of his spare time constructing simple microscopes composed of double convex glass lenses held between two silver plates (figure 1.10b). His microscopes could magnify about 50 to 300 times, and he may have illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at a 45-degree angle to wil11886_ch01_001-019.indd 11 the specimen plane. This would have provided a form of darkfield illumination whereby organisms appeared as bright objects against a dark background. Beginning in 1673, Leeuwenhoek sent detailed letters describing his discoveries to the Royal Society of London. It is clear from his descriptions that he saw both bacteria and protists (figure 1.10c). Darkfield microscope: bright object, dark background (section 2.2) Culture-Based Methods for Studying Microorganisms Were a Major Development As important as Leeuwenhoek’s observations were, the development of microbiology essentially languished for the next 200 years until techniques for isolating and culturing microbes in the laboratory were formulated. Many of these techniques were developed as scientists grappled with the conflict over the theory of spontaneous generation. This conflict and the subsequent studies on the role played by microorganisms in causing disease ultimately led to what is now called the golden age of microbiology. Spontaneous Generation From earliest times, people had believed in spontaneous generation—that living organisms could develop from nonliving matter. This view finally was challenged by the Italian physician Francesco Redi (1626–1697), who carried out a series of experiments on decaying meat, which was thought to produce maggots spontaneously. Using covered and uncovered containers of meat, Redi clearly demonstrated that maggots on decaying meat resulted from the presence of fly eggs, and meat did not spontaneously generate maggots. Other experiments helped discredit the theory for larger organisms. However, Leeuwenhoek’s communications on microorganisms renewed the controversy. Some proposed that microbes arose by spontaneous generation but larger organisms did not. They pointed out that boiled extracts of hay or meat gave rise to microorganisms after sitting for a while. In 1748 the English priest John Needham (1713–1781) suggested that the organic matter in these extracts contained a “vital force” that could confer the properties of life on nonliving matter. A few years after Needham’s experiments, the Italian priest and naturalist Lazzaro Spallanzani (1729–1799) sealed glass flasks that contained water and seeds and then placed the flasks in boiling water for about 45 minutes. He found that no growth took place as long as the flasks remained sealed. He proposed that air carried germs to the culture medium but also commented that external air might be required for growth of animals already in the medium. The supporters of spontaneous generation responded that heating the air in sealed flasks destroyed its ability to support life, and therefore did not discredit the theory of spontaneous generation. In the mid-1800s, several investigators attempted to counter such arguments. These experiments involved allowing air to enter a flask containing a nutrient solution after boiling. The air was either also very hot or it was filtered through sterile cotton wool. In all cases, no microbial growth occurred in the medium. Despite 23/10/18 9:23 am 12 CHAPTER 1 | The Evolution of Microorganisms and Microbiology 1668 Redi refutes spontaneous generation of maggots. 1665 Hooke publishes Micrographia. 1674–1676 Leeuwenhoek discovers “animalcules.” 1543 Publication of Copernicus’s work on heliocentric solar system 1620 Francis Bacon argues for importance of inductive reasoning in scientific method. 1876 Bell invents telephone. 1903 Wright brothers’ first powered aircraft 1914 World War I begins. 1911 Rous discovers a virus can cause cancer. 1915–1917 D’Herelle and Twort discover bacterial viruses. 1923 First edition of Bergey’s Manual 1917 Russian Revolution 1918 Influenza pandemic kills over 50 million people. 1939 World War II begins. 1937 Krebs discovers citric acid cycle. 1945 Atomic bomb dropped on Hiroshima. 1950 Korean War begins. 1884 Koch’s postulates published; Metchnikoff describes phagocytosis; autoclave developed; Gram stain developed. 1885 Pasteur develops rabies vaccine. 1899 Beijerinck proves virus causes tobacco mosaic disease. 1900 Planck develops quantum theory. 1908 First Model T Ford 1876 Koch demonstrates that Bacillus anthracis causes anthrax. 1888 Beijerinck isolates root nodule bacteria. 1898 SpanishAmerican War 1905 Einstein’s theory of relativity 1861 Pasteur disproves spontaneous generation. 1861–1865 American Civil War 1887–1890 Winogradsky studies sulfur and nitrifying bacteria. 1893 Munsch paints The Scream. 1798 Jenner introduces cowpox vaccination for smallpox. 1854 Snow traces cholera source to water pump. 1775 American Revolution begins. 1859 Darwin’s Origin of Species 1687 Newton’s Principia published. 1879 Edison’s first light bulb 1889 Eiffel Tower completed. 1765–1776 Spallanzani attacks spontaneous generation. 1927 Lindbergh’s transAtlantic flight 1933 Hitler 1929 Stock becomes market crash chancellor of Germany. 1953 Watson and Crick propose DNA double helix. 1928 Griffith discovers bacterial transformation. 1929 Fleming discovers penicillin. 1932 Knoll and Ruska build first electron microscope. 1990 First human gene therapy testing begun. 1961 Jacob and Monod propose lac operon. 1961 First human in space 1969 Neil Armstrong walks on the moon. 1983–1984 HIV isolated and identified by Gallo 1970 Arber and Smith and Montagnier; discover restriction Mullis develops endonucleases. 1992 First PCR technique. human trials 1977 Woese divides of antisense prokaryotes into therapy Bacteria and Archaea. 1973 Vietnam War ends. 1980 First home computers 1981 First space shuttle launch Figure 1.9 Some Important Events in the Development of Microbiology. Milestones in microbiology are marked in red; other historical events are in black. wil11886_ch01_001-019.indd 12 1991 Soviet Union collapses. 2001 World Trade Center attack; Anthrax bioterrorism attacks in U.S. 2010 First bacterium with synthetic genome constructed. 2005 Genome of 1918 influenza virus sequenced. 2003 Second war with Iraq; SARS outbreak in China 2014 2-year Ebola outbreak 2010 H1N1 influenza outbreak 6/13/19 12:27 PM 1.3 Microbiology Advanced as New Tools for Studying Microbes Were Developed 13 these experiments, the French naturalist Felix Pouchet (1800–1872) claimed in 1859 to have carried out experiments conclusively proving that microbial growth could occur without contact with air. Pouchet’s claim provoked Louis Pasteur (1822–1895) to settle the matter of spontaneous generation. Pasteur (figure 1.11) first filtered air through cotton and found that objects resembling plant spores had been trapped. If a piece of the cotton was placed Figure 1.11 Louis Pasteur. in sterile medium after air ©Pixtal/age fotostock had been filtered through it, microbial growth occurred. Next he placed nutrient solutions in flasks, heated their necks in a flame, and pulled them into a variety of curves. The swan-neck flasks that he produced in this way had necks open to the atmosphere (figure 1.12). Pasteur then boiled the solutions for a few minutes and allowed them to cool. No growth took place even though the contents of the flasks were exposed to the air (figure 1.12). Pasteur inferred that growth did not occur because dust and germs had been trapped on the walls of the curved necks. If the necks were broken, growth commenced immediately. Pasteur had not only resolved the controversy by 1861 but also had shown how to keep solutions sterile. The English physicist John Tyndall (1820–1893) and the German botanist Ferdinand Cohn (1828–1898) dealt a final blow to spontaneous generation. In 1877 Tyndall demonstrated that dust (a) Lens Specimen holder Focus screw Handle (b) Microbes being destroyed Figure 1.10 Antony van Leeuwenhoek. (a) An oil painting of Leeuwenhoek. (b) A brass replica of the Leeuwenhoek microscope. Inset photo shows how it is held. (c) Leeuwenhoek’s drawings of bacteria from the human mouth. (c) wil11886_ch01_001-019.indd 13 (a) ©Bettmann/Getty Images; (b) ©Kathy Park Talaro/Pasadena City College; (c) ©Dr. Jeremy Byrgess/SPL/Getty Images Vigorous heat is applied. Broth free of live cells (sterile) Neck on second sterile flask is broken; growth occurs. Neck intact; airborne microbes are trapped at base, and broth is sterile. Figure 1.12 Pasteur’s Experiments with Swan-Neck Flasks. 23/10/18 9:23 am 14 CHAPTER 1 | The Evolution of Microorganisms and Microbiology did indeed carry germs and that if dust was absent, broth remained sterile even if directly exposed to air. During the course of his studies, Tyndall provided evidence for the existence of exceptionally heat-resistant forms of bacteria. Working independently, Cohn discovered that the heat-resistant bacteria recognized by Tyndall were species capable of producing bacterial endospores. Cohn later played an instrumental role in establishing a classification system for bacteria based on their morphology and physiology. Bacterial endospores are a survival strategy (section 3.9) These early microbiologists not only disproved spontaneous generation but also contributed to the rebirth of microbiology. They developed liquid media and the methods for sterilizing it so that microbes could be cultured. These techniques were next applied to understanding the role of microorganisms in disease. Microorganisms and Disease For hundreds of years, most people believed that disease was caused by supernatural forces, poisonous vapors, and imbalances among the four humors thought to be present in the body. The role of the four humors (blood, phlegm, yellow bile [choler], and black bile [melancholy]) in disease had been widely accepted since the time of the Greek physician Galen (129–199). Support for the idea that microorganisms cause disease—that is, the germ theory of disease—began to accumulate in the early nineteenth century from diverse fields. Agostino Bassi (1773– 1856) demonstrated in 1835 that a silkworm disease was due to a fungal infection. In 1845 M. J. Berkeley (1803–1889) proved that the great potato blight of Ireland was caused by a water mold (then thought to be a fungus), and in 1853 Heinrich de Bary (1831–1888) showed that fungi caused cereal crop diseases. Pasteur also contributed to this area of research in several ways. His contributions began in what may seem an unlikely way. Pasteur was trained as a chemist and spent many years studying the alcoholic fermentations that yield ethanol and are used in the production of wine and other alcoholic beverages. When he began his work, the leading chemists were convinced that fermentation was due to a chemical instability that degraded the sugars in grape juice and other substances to alcohol. Pasteur did not agree; he believed that fermentations were carried out by living organisms. In 1856 M. Bigo, an industrialist in Lille, France, where Pasteur worked, requested Pasteur’s assistance. His business produced ethanol from the fermentation of beet sugars, and the alcohol yields had recently declined and the product had become sour. Pasteur discovered that the fermentation was failing because the yeast normally responsible for alcohol formation had been replaced by bacteria that produced acid rather than ethanol. In solving this practical problem, Pasteur demonstrated that fermentations were due to the activities of specific yeasts and bacteria, and he published several papers on fermentation between 1857 and 1860. Pasteur was also called upon by the wine industry in France for help. For several years, poor-quality wines had been produced. Pasteur referred to the wines as diseased and demonstrated that particular wine diseases were linked to particular microbes contaminating the wine. He eventually suggested a wil11886_ch01_001-019.indd 14 method for heating the wines to destroy the undesirable microbes. The process is now called pasteurization. Indirect evidence for the germ theory of disease came from the work of the English surgeon Joseph Lister (1827–1912) on the prevention of wound infections. Lister, impressed with Pasteur’s studies on fermentation, developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds. Instruments were heat sterilized, and phenol was used on surgical dressings and at times sprayed over the surgic