Fundamentals in Biology 1: From Molecules to the Biochemistry of the Cell PDF

Department of Biology Fundamentals in Biology 1: From Molecules to the Biochemistry of the Cell 1 Chapter 1: Evolving life Julia Vorholt "Nothing in biology makes sense except in the light of evolution." (T. Dobzhansky) Diversity and Unity of Life...

Department of Biology Fundamentals in Biology 1: From Molecules to the Biochemistry of the Cell 1 Chapter 1: Evolving life Julia Vorholt "Nothing in biology makes sense except in the light of evolution." (T. Dobzhansky) Diversity and Unity of Life Life on Earth is immensely diverse (Figure 1.1). Current estimates for the number of species range from 2 to 100 million or more. At first glance, squid that fluoresce in the oceans may seem to have nothing in common with microscopic yeast used for brewing beer, or with giant sequoias that can grow up to 80 meters high in some forests in North America. Nonetheless, all living forms share important fundamental traits and descend from a common origin. From this evolutionary perspective, all organisms are related to each other, and all organisms consist of one or many cells as organizational units. Moreover, no life form exists entirely on its own: Every species and every single organism depends in one way or another on interactions with both abiotic (non-living) and biotic (living) factors in the environment. All these relationships are essential to the very nature of life. In this chapter we will illustrate some of these interactions and how they have shaped the geological history of Earth, resulting in the diversity and composition of the biomass of the various life forms that exist to this day. Figure 1.1. A selection of life forms. The photographs show a variety of organisms: (A) Praying mantis (top left); (B) Witches’ butter fungus; (C) White rhinoceros; (D) Rhinoceros beetle; (E), Diatom algae; (F) Giant sequoia trees. Image credits: Shutterstock ID 1815508841, 494449975, 312709391, 773915281, 2100233800, 1100584175. 2 Symbiosis – living together When we breathe, we consume molecular oxygen (O2) that was produced by photosynthesis, so we indirectly interact with plants, algae, and phototrophic cyanobacteria. But there are also direct, short- and long-term interactions, referred to as symbioses, from the Greek word συμβίωσις for "living together". An interaction between organisms can be beneficial to one or more of the partners (a positive, mutualistic interaction), harmful to one or more of the organisms involved (a negative, antagonistic interaction), or have no substantial effect (neutral interaction). Such symbiotic associations can be obligatory or facultative, depending on whether the interaction is crucial for survival in a given environment or not. In ecology, which concerns the interactions between organisms and interactions of organisms with their environment, different terms are used to describe various types of relationships among organisms such as parasitism and mutualism. In parasitic relationships, one partner benefits from the interaction while the other is harmed. An example of such an interaction is ticks, which feed on the blood of mammals, including Figure 1.2. Examples of mutualistic symbiosis. The humans. They can transmit bacteria that cause images illustrate examples of mutualistic symbioses, a type of symbiotic relationship where both partners Lyme disease and harm the host. Conversely, benefit from the interaction: (A) Soybean root nodules mutualistic relationships provide benefits to both hosting nitrogen-fixing bacteria; (B) Lichens, a symbiosis of fungi and photosynthetic algae or partners. An ancient example of mutualism are cyanobacteria, growing on rocks; (C) Corals with lichens, fungi that are associated with photosynthetic dinoflagellates providing carbohydrates in exchange for carbon dioxide, nutrients, and a phototrophic bacteria or algae. Another protected environment; (D) Bees and flowering plants, beneficial partnering is between plants and where bees obtain nectar and pollen and, in return, pollinate the plants, aiding in reproduction. Image certain fungi, the mycorrhiza, which supply plant credits: Images credits: Shutterstock ID 345198950, 1730633506, 1264014256, 1432463195. roots with nutrients from soil, in particular phosphorus, and benefit from the plant’s photosynthesis. Plant interactions with mycorrhizal fungi have been evolving since plants first colonized land over 400 million years ago and are widespread today. Other examples of mutualistic relationships are those between nitrogen-fixing bacteria and leguminous plants such as soybean or pea plants, corals which are animals that associate with unicellular phototrophs, and the relationship between pollinators and flowering plants (Figure 1.2). Yet another example of a mutualistic relationship involves human beings and the community of microorganisms residing in and on our bodies: the human microbiome. The largest part of this microbiome is found in the gastro-intestinal tract, where roughly 10,000,000,000,000 (10 trillion) microbial cells produce vitamins and provide nutrients by digesting food that humans consume. The microbiome interacts with humans in many other ways but, despite containing approximately as many cells as the human body, accounts for only one percent of its weight. This is because microbial cells are much smaller than an average human cell: Rod-shaped bacteria are typically 2 µm long and 1 µm thick (50 times smaller than the diameter of a human hair). Contrary to their minuscule size, however, their ecological importance is huge. 3 Microorganisms are the main drivers of global biogeochemical cycles and can be found everywhere: from hot springs to glaciers, as well as in association with virtually all multicellular plants and animals. Composition of the contemporary living world Before addressing the more fundamental questions on how to define life, let us take a look at the abundance of different life forms around the globe today. Scientists have compiled data from various sources and an incredible number of samples, using techniques including satellite remote sensing, counting by eye or using microscopes, to modern techniques such as next- generation sequencing. Based on the collected information and extrapolations, they estimated the total biomass on Earth, represented as the mass of carbon, a common proxy value that is independent of water content. They concluded that the biomass across all taxonomic groups adds up to 550,000,000,000 (550 billion) tons of carbon (C), of which 82% belongs to plants (Figure 1.3). The second largest group in terms of biomass is bacteria, which account for about 13% of the total carbon in living organisms. Other organisms include archaea, fungi, protists (a diverse group of mostly unicellular eukaryotic organisms), and animals, which together add up to about 5% of the global biomass. Figure 1.3. Biomass composition in the living world. (Left) Global biomass distribution is represented graphically as a square showing the fraction of taxonomic groups relative to Earth's total estimated biomass (carbon fraction). Total biomass (carbon) on Earth is approximately 550 Gt. (Right) Biomass distribution of different animal taxa, excluding low biomass estimates for reptiles and amphibians. Data are replotted from Bar-On et al. 2018 PNAS 115: 6506-6511. Focusing on the biomass of animals (which makes up 2 billion out of the 550 billion tons of carbon) reveals that humans and livestock such as cattle and pigs far outweigh the biomass of all wild mammals at present. Modern humans (Homo sapiens) originated about 200,000 years ago and have contributed to a five-fold reduction in the biomass of wild animals since the expansion of agriculture and human population about 12,000 years ago, and especially since the Industrial Revolution in the late 18th century. If the entire Earth’s history were compressed into 24 hours, this reduction in biomass would have occurred in less than a second. While abrupt changes did occur in the past on Earth (think about the extinction of the 4 dinosaurs), this is the first time that a single species, i.e. humans, changed the Earth in such short time. Apart from the biomass distribution on Earth, the estimated number of living organisms in different taxonomic groups can be correlated. Bacteria and archaea were the first organisms to have emerged and survived on Earth, probably almost 3.8 billion years ago, and still dominate the world in terms of abundance. They are invisible to the naked eye and have been referred to as "the unseen majority". It seems that our planet has always been, and perhaps will always be, in the age of microorganisms. Bacteria alone comprise an estimated 1030 individuals, making them the most numerous organisms on Earth. Viruses also exist as biomass entities and outnumber even bacteria, but they are not considered living organisms, as we will see later. Plotting the overall biomass of the various groups against the number of organisms (Figure 1.4) shows a correlation, the higher the number of individuals the higher the total biomass. However, there is an exception to this rule: plants. Their biomass is higher compared to that of other organisms like fish or insects. This is because they can grow to enormous sizes. For example, one single giant sequoia tree can reach a weight up to four thousand tons. Figure 1.4. Relationship between abundance and biomass. The plot shows the total estimated number of individuals in each taxon versus the total estimated biomass of the same taxon, with error bars indicating the uncertainty of biomass estimates. In general, taxonomic groups with a higher number of individuals (circles) have a higher biomass. Plants diverge from other taxonomic groups, as individual plants can produce a large biomass. Data are reblotted from Bar-On et al. 2018 PNAS 115: 6506-6511. 5 How are all the different forms of life distributed across the various environments on Earth? Plant and fungal biomass is primarily concentrated in terrestrial ecosystems, while most protist and animal biomass is located in marine environments. Bacteria and archaea, on the other hand, are mainly found in deep subsurface environments, such as subseafloor sediments, the oceanic crust, and terrestrial aquifers (Figure 1.5). Let us return to the overall biomass distribution on Earth, now not viewed by major groups of organisms, but by ecosystems at large. Terrestrial ecosystems host the largest share of the Earth’s total biomass (Figure 1.5). In contrast, marine biomass is almost a hundred times smaller, even though oceans cover 71% of the Earth’s surface. Despite the large difference in biomass on land and in the oceans, the primary productivity—the amount of carbon captured from carbon dioxide (CO2) by photosynthesizers per year—is roughly equal in both environments (Figure 1.5). As a result, CO2 conversion per biomass is different on land compared to in the oceans, where the biomass that is formed is degraded at much higher rates as we will discuss in Chapter 8. Figure 1.5. Biomass distribution across different environments and taxonomic groups on Earth. (A) Proportion of major groups per ecosystem: terrestrial (beige), marine (blue) and deep subsurface (gray) (deep subsurface is defined as the marine subseafloor sediment and oceanic crust in addition to terrestrial ecosystems deeper than 8 m without soil). Plants and fungi are primarily terrestrial, protists and animals are mostly marine, and bacteria and archaea are abundant in deep subsurface environments. (B) Total biomass estimates per ecosystem highlight the predominance of terrestrial systems. In contrast, marine systems account for only about 1% of the total biomass. Terrestrial systems have a high fraction of primary producers, autotrophs that make their organic molecules from CO2 (green bar), compared to consumers, which are heterotrophs (red bar). Conversely, the fraction of primary producers is lower in marine systems compared to consumers. Primary producers in marine systems have a rapid turnover of biomass. The global primary productivity, the amount of organic material fixed from CO2, is about 50% in terrestrial and marine systems (about 50 Pg carbon per year in total). Panels A and B are replotted from data presented in Bar-On et al. 2018 PNAS 115: 6506-6511. 6 Biology = Physics + Chemistry + Evolution This simple equation is not a mathematical formula, but a reminder that biology, the study of life, is based on principles from physics, chemistry, and evolution. Physics and chemistry provide a fundamental understanding of the processes that occur in living organisms. Physics, as the most fundamental discipline, informs biology with its mathematical descriptions of natural phenomena, such as the properties of complex systems and the laws of thermodynamics. Chemistry deals more specifically with the properties, structure, and reactions of the elements and molecules that constitute all matter, living or not. In addition to foundational rules from physics and chemistry, biology is concerned with the study of living organisms and how life forms changed over time to give rise to the diversity we see today. The importance of evolutionary thinking in biology cannot be overstated, as famously put by the geneticist Theodosius Dobzhansky: "Nothing in biology makes sense except in the light of evolution." It is not by chance that the chemistry of life is based on carbon (C), an element on Earth that can form long chains as well as branched and cyclic compounds that are both stable and flexible, as we will see in Chapter 2. Carbon is the central element of organic chemistry because it readily forms bonds with many different elements (H, N, O, P, S, and various metals), but also with other carbon atoms, enabling the generation of diverse molecular structures that allowed the emergence of life. Throughout the course of life's history, organisms have harnessed their capacity to produce increasingly complex organic molecules. All biomolecules within cells contain carbon, which accounts for half of the biomass dry weight, serving as a unit of biomass on Earth, as shown in Figure 1.3. According to the second law of thermodynamics (Box 1.1), every system tends to maximize its entropy, a measure of probability, to reach its thermodynamic equilibrium. Thus, an isolated system changes from a state of lower probability to one of higher probability, or in more illustrative terms, from "ordered" to "disordered". But living systems maintain matter in a highly ordered state, therefore seeming to contravene the second law of thermodynamics. The ordered nature of living beings stems from the fact that they do not live in isolation but convert energy that they obtain from their environment. If this energy is not available or cannot be used for some reason, the organism’s internal structure moves towards equilibrium and higher disorder. This holds true for organisms as such but manifests also in biochemical reactions that run within cells. Reactions that occur with a net release of free energy are termed exergonic, they move in the direction of higher probability. In contrast, endergonic reactions must be forced towards lower probability, such as those required for the biosynthesis of proteins or the polymerization of the genetic material DNA. This is achieved by coupling endergonic to exergonic reactions, as we will discuss in subsequent chapters. Importantly, this coupling characterizes the living state: a state of disequilibrium that from a thermodynamical point of view is extremely improbable. For life to function, this imbalance must be constantly maintained, illustrating that living systems must be open systems that continuously exchange energy and matter with the environment. Changes in free energy (work, ΔG), the relevant form of energy in biological systems that operate under isothermal conditions, allow quantitative predictions about energy flow at the level of individual reactions, cells, communities, and the ecosystem, that is, activities of organisms in context. 7 Box 1.1. The laws of thermodynamics Energy can be transferred in two ways: as work or as heat. Work involves an ordered, directed process, while heat refers to the disordered transfer of energy, such as the random motion of particles. The first law: In an isolated system, energy cannot be created or destroyed, it can only be transformed (work + heat = constant) The second law: The state of equilibrium that systems tend to is the one that maximizes disorder (entropy). This is a consequence of the fact that under isothermal conditions, work can be converted to heat but not vice versa (work → heat). In chemical reactions, the change in Gibbs free energy (ΔG) is used to indicate the maximum amount of work that can be performed. The maximal amount of heat change associated with chemical reactions is referred to as the change in free enthalpy (∆H). In contrast to mechanical systems, chemical reactions require a correction that accounts for the different heat capacities of substrates and products: ∆G = ∆H - T∆S, where ∆S (entropy change) represents the different heat capacities (Q/T) and T the absolute temperature. Biological systems generally proceed at roughly constant temperature, pressure and volume, so that the free energy change ΔG rather than the free enthalpy change ΔH is of importance. What is Life? It is anything but trivial to define life, and there are many ways of approaching the problem. In 1944, the physicist Erwin Schrödinger published a famous essay called "What is life?" in which he describes life processes in physicochemical and thermodynamic terms. He speculated that the hereditary material would "contain in some kind of code-script the entire pattern of the individual's future development and of its functioning in the mature state". More recently, John F. Allen and colleagues noted that life "is the harnessing of chemical energy in such a way that the energy-harnessing device makes a copy of itself." The interaction with the environment to acquire energy and nutrients, as well as the ability to reproduce, are fundamental characteristics that constitute essential properties of life. Centuries-long discussions have not resolved the question about the universal features of life, nor is there a generally accepted definition for life. This is already evident when looking at the question of where and when life on Earth originated. Theoretical considerations and seminal experiments have enriched our current ideas of how life may have emerged from geochemical processes that allowed non-living materials to accumulate and interact with each other, but we cannot determine the exact point at which this prebiotic evolution crossed the threshold to life (Figure 1.6). The appearance of living matter must be considered the major and decisive innovation in Earth history, which, with the subsequent major innovations in life's history, ultimately profoundly affected the condition of our planet. 8  Figure 1.6. Simplified scheme illustrating the increasing complexity in biology. Geochemical processes led to the accumulation of organic molecules that formed more complex systems, which crossed the origin of life approximately 4 billion years ago. Systems innovations were maintained and built upon over time, illustrated by a purple line. Easier than defining life, it seems, is describing life and its key components, which relate to the characteristics we introduced above and can relate to current knowledge about cellular molecules and biochemistry that exist today. Most scientists agree with the view that an organism must fulfill four main criteria to be alive (Figure 1.7): First, an organism must have ways to convert and use energy to maintain itself, to grow, and to divide. As we will see later, energy is not just consumed or released during biochemical reactions, it can also be stored spatially and sustained in different forms such as electrochemical gradients in cellular compartments, where separating membranes act as barriers. Membranes separate the inside of the cell from its environment, a second criterion critical to life. Figure 1.7. Major criteria that define life. Living organisms share four criteria. They: 1) harness energy from their environment; 2) separate inside from outside with membranes, forming cells; 3) pass information on to offspring; and 4) evolve according to Darwinian principles. Energy and nutrients are essential for building new cell biomass, with cell proliferation being an autocatalytic process. 9 Living organisms must also be able to inherit instructions for replication to their offspring. The "code-script" as Schrödinger termed it, turned out to be the sequence of building blocks (nucleotides) in the nucleic acid DNA that makes up the genome of an organism. Cells as the fundamental unit of organisms replicate this genetic information before they divide and multiply. The instructions for growth and replication are encoded in this hereditary material. It ensures that "the copy of itself" is faithfully reproduced in an autocatalytic process. But during this process, changes in the form of mutations may occur, for example, leading to altered properties or quantities of proteins, among other changes. Given enough generations, such changes in heritable information allow populations of organisms to adapt to changing conditions in their environment or to expand into new environments. This pinpoints a fourth criterion of all living systems, the ability to evolve. There is no life without heritable variation exposed to natural selection – the principles of Darwinian evolution. These key properties of life also guide the structure of this book, which focuses on core processes fundamental to every cell (Figure 1.8). All life forms share common molecular Figure 1.8. Core features of a cell. A cell, the smallest unit of life, shares universal biochemical features across features and rely on the same kinds of all life forms on Earth. A membrane separates inside and information-containing macromolecules, that outside, and central metabolic reactions occur within the cell (cytosol), requiring numerous catalysts. The catalysts is, polymers such as DNA, RNA, and proteins. are also necessary for copying genomes and generating These are in turn built from a common set of new catalysts, resulting in an autocatalytic biological molecular precursors as we will discuss in system. Chapter 2. Key points: ▪ Organisms can interact with each other in various ways; this can lead to symbiosis. ▪ Plants dominate the biomass on Earth, while bacteria predominate in terms of the number of living organisms. ▪ Life is characterized by the ability to harness energy, assemble macromolecules from nutrients taken up from the environment, reproduce, and evolve. Questions 1) Cold air has less entropy than hot air. The second law of thermodynamics states that entropy always increases. Does a refrigerator violate this law? 2) Where does the energy that is needed to drive the polymerization of macromolecules such as DNA come from? 10 Earth history and evolution of life on Earth The evolution of life on Earth is part of the 4.57 billion-year history of the planet (Box 1.2). The diversity of modern life forms is the result of interactions between organisms and their interactions with the environment (Figure 1.9). While Earth provided the Figure 1.9. Earth-life feedback loops. Illustration of the fundamental conditions for life to arise, such reciprocal feedback between Earth and life: Earth provides as water and essential elements, life in turn matter and energy for life to evolve, while life shapes the geology of the planet. Since the onset of photosynthesis, has also modified the geochemistry of the light energy is also used. planet. A prime example of impact of life on Earth occurred about halfway through its history, marked by the emergence of molecular oxygen (O2). This development profoundly Figure 1.10. Banded Iron Formation (BIF). Alternating layers of sedimentary rock containing iron(Fe)-rich changed Earth's geochemistry. The impressive minerals formed approximately 2.4 billion years ago. The iron layers in rocks (Figure 1.10) are testimony red color is due to hematite (Fe2O3), a mineral produced by the oxidation of ferrous iron (Fe2+) during erosion. of this change,, brought about by reaction of O2 During this period, cyanobacteria began to perform with soluble reduced iron (Fe2+) and photosynthesis, releasing molecular oxygen (O2) as a subsequent precipitation of iron as Fe3+. byproduct. This O2 reacted with dissolved ferrous iron in the ocean, oxidizing it to ferric iron (Fe3+), resulting in Additionally, the increased O2 concentrations layers that represent ancient geological records. triggered dramatic transformations in the forms Location: Karijini National Park, Pilbara, Australia (Image of life on Earth, as discussed below. credit: Shutterstock ID 698776915, 2037404732). Box 1.2. The planetary setting for the development of life In this box, we will cover the chronology of Earth’s history and some of the requirements for life to emerge. The age of the Earth and how we know it The Earth is approximately 4.57 billion years old 4,570,000,000 years old 4.57x109 or 4.57 Gyr or 4.57 Ga years old (Giga is a prefix for 109) Scientists use various methods to determine the age of the Earth, one of the most widely accepted being the principle of radioactive decay in certain minerals. A mineral that is found in many rocks is zircon (ZrSiO4). Zircon, which contains the element zirconium (Zr), forms in the magma of volcanoes. The mineral is dispersed as ash after an eruption and can be trapped as a volcanic layer when rocks form. Zircon, like many other minerals, is not pure. The element uranium (U) sometimes substitute for Zr in zircon due to its similar size. Uranium is useful for dating the Earth: It decays to another element, lead (Pb), at a fixed rate and it does so very 11 slowly. This allows researchers to determine how much time has passed since the rock was formed by measuring its lead content. Nevertheless, because Earth has been and continues to be geologically active, with heat from its interior causing rocks to form and be destroyed, no parts of Earth that date back to its origin remain. Crucial for dating the age of the Earth are meteorites that formed elsewhere in our Solar System, specifically in the asteroid belt between Mars and Jupiter, and happened to fall on the planet. These meteorites stem from bodies that cooled and solidified more quickly than the Earth, preserving their more ancient mineral makeup. Scientists have determined that the oldest meteorite found is 4.567 billion years old. This information, along with other estimates, suggests that the Earth is slightly older than this date. The presence of water and elements essential for life Life as we know it depends on essential elements, including hydrogen (H) and oxygen (O), which make up water and are also components of biomolecules. Other elements, in particular carbon, nitrogen, phosphorus, and sulfur are also crucial for the existence of life. It is widely believed that stars and planets form by accretion through the gravitational collapse of interstellar clouds of gas and dust, which first form a rotating disk and then develop into discrete bodies, with a star at the center and planets orbiting it. Earth, unlike Jupiter, is too small to retain elements such as hydrogen, oxygen, carbon, nitrogen, phosphorous, and sulfur due to too low gravitational forces. It was concluded that these elements were delivered to Earth by the collision of water-rich meteorites early in its history. The relevance of water lies in its composition (hydrogen and oxygen) and its role as a solvent, for carbon, nitrogen, phosphorous and sulfur-containing compounds. There is evidence that Earth collided with another celestial body, known as Theia, which caused the planet to melt and led to the formation of the Moon. It is believed that while the Earth was still in a molten state, its first oceans and its atmosphere began to form as a result of degassing from magma. It is estimated that an ocean existed on Earth as early as 4.4 billion years ago. Long-term habitability of Earth Life required a very long time to evolve to the diversity we observe today on Earth (Figure 1.3), the only planet where we know that life exists. This implies that life required a relatively stable environment over all this time of continuous history. It does not mean that the surface environment was always hospitable for life. There have been several catastrophic events that wiped out almost all marine or terrestrial species living at the time, such as the dinosaurs 66 million years ago due to an asteroid impact. Currently, human actions are having a substantial impact on Earth, leading to climate change and a loss in biodiversity. The Sun is the primary source of energy that powers all life on the surface of Earth. Based on Earth's distance from the Sun, an atmosphere consisting of more than 99% N2 and O2 that does not absorb infrared light, and a planetary albedo, the fraction of how much sunlight a planet reflects back into space (0 no reflection, 1 complete reflection), of approximately 0.3, we would expect the average temperature to be about -18°C (255K) and water to be frozen. The fact that the global and annual average is 15°C is due to the presence of small amounts of greenhouse gases in our atmosphere, primarily carbon dioxide (CO2), which ultimately comes from the interior of the Earth, mainly through volcanic magma. Note that at higher temperature water vapor becomes more abundant in the atmosphere and contributes substantially to the natural greenhouse effect by absorbing and re-emitting infrared radiation. 12 If CO2 were continuously being added to the atmosphere, it would build up and lead to a gradual increase in temperature. However, there are also processes that remove CO2 from the atmosphere. One such process is chemical weathering, which operates over vast geological timescales of thousands to millions of years. It has occurred naturally since before life existed on Earth and continues to do so today. In this process, carbonate (CO32-) or bicarbonate (HCO3-), which form from CO2 and H2O, react with cations such as calcium (Ca2+) to form the carbonate mineral calcite, also known as limestone. Another process that removes CO2 from the atmosphere is carbon dioxide fixation by living organisms (a process we will discuss in Chapter 7). However, only a tiny fraction of the organic carbon fixed in this way today is sequestered for long periods of time. This is in contrast to times far back in Earth's history, when plants first emerged and gave rise to deposits that we now refer to as coal and methane, fossil fuels produced by compression of organic plant matter in terrestrial systems - or the remains of ancient marine plants and animals in marine systems, which led to the formation of oil (petroleum) and methane. The fraction of CO2 in the atmosphere in the pre-industrial period was 0.00028, which means that for every million molecules of air there are 280 molecules CO2 (280 parts per million, ppm). This small amount of CO2 is crucial to the surface conditions of our planet. As of 2024, this amount has increased to 423 ppm, corresponding to an increase of more than 50%, leading to global warming and climate change. Relative to the total age of Earth, life emerged rather early, approximately half a billion years into its history or around 4 billion years ago (Figure 1.11). For nearly half of the history of life on Earth, only bacteria and archaea existed. These two groups of organisms are collectively referred to as "prokaryotic", a term derived from ancient Greek, "pro" meaning "before" and "karyon" meaning "nut" or "kernel", alluding to the nucleus in eukaryotes (“true nut”), the intracellular compartment containing DNA. Bacteria and archaea were, and continue to be, relatively simply organized, typically existing as single cells that lack specialized membranous compartments such as a nucleus or organelles. In Chapter 6, we will discuss the extraordinary metabolic processes that prokaryotes evolved to extract energy from their surroundings. The first cells harnessed energy through chemosynthesis, exploiting the chemical reactions of compounds in their environment. Later, cells evolved protein complexes known as photosystems that enabled conversion of sunlight into chemical energy. Oxygenic photosynthesis stands out among the different energy- harvesting mechanisms, as bacteria making their living with light energy conversion found a way to split off electrons from water and liberate molecular oxygen (O2) as a byproduct. Starting around 2.4 billion years ago, the metabolic activities of these bacteria resulted in the significant accumulation of O2 in the atmosphere. This phenomenon, known as the Great Oxidation Event, catalyzed a profound shift in atmospheric chemistry and dramatically altered the appearance of our planet (Figure 1.11). This transformation had two important consequences. First, O2 in the atmosphere reacted to yield ozone (O3) in the upper atmosphere which protects the Earth’s surface from dangerous ultraviolet (UV) light from the Sun. Without an ozone layer, life outside the ocean would probably have been impossible. Second, and arguably more fundamental, the accumulation of O2 paved the way for the evolution of aerobic metabolism, which is highly rewarding from a bioenergetic standpoint as 13 we will see in Chapter 6. Molecular oxygen used by many eukaryotes traces back to the common ancestor of eukaryotic cells, which emerged around 2 billion years ago. The ability to respire with O2 is also linked to the emergence of life on land. Complex multicellular eukaryotes left the oceans and began the colonization of terrestrial habitats – first fungi, then plants, then animals – starting around 500 million years ago. The first modern humans appeared much later, only about 230,000 years ago. The transition to life on land marks the transition to life in permanently oxic environments. It is now assumed that the emergence of land plants was the cause of the high O2 content in the atmosphere (21% [v/v]) that we see today. Figure 1.11. History of major biological innovations on Earth. Bacteria and Archaea date back to the origin of cellular life, eukarya evolved about 2 billion years ago (BYA). The Great Oxidation Event caused by oxygenic photosynthesis led to the accumulation of molecular oxygen. Before this, the atmosphere and oceans were anoxic. Multicellular organisms diversified substantially during the Cambrian explosion about (~0.5 BYA). 14 Three domains of Life The evolutionary history of life on Earth (Figure 1.11) can be contextualized by considering the three currently accepted domains of life: Bacteria, Archaea, and Eukarya. As previously noted, Eukarya are relatively young evolutionarily compared to the two prokaryotic groups, Bacteria and Archaea, and arose from a symbiosis of bacterial and archaeal cells. The last universal common ancestor (LUCA) of all cells is a theoretical concept in evolutionary biology based on the biochemical properties shared among all existing organisms (allowing for losses in some specialized parasitic forms). LUCA thus implies a common origin of cellular life from which all other forms of life diversified. It is sometimes depicted as a hypothetical single-celled organism. However, we do not know that such a cell existed, and it may be misleading to think of a singular organism as a physical LUCA entity. An alternative, broader, and more probable scenario is that LUCA was a population of protocells that exchanged genetic material and possessed a number of characteristics, such as the ability to store energy in concentration gradients across membranes, transcribe genomic information, i.e., DNA (or RNA) into RNA, and translate RNA into proteins (Chapter 4). These basic biological macromolecules, which formed from simpler building blocks, further detailed in Chapter 2, in the distant past, are still present today, universally, in all living organisms, regardless of whether they are pro- or eukaryotic. The central functions common to all cells can be seen as a kind of molecular fossil, a testimony of the shared evolutionary history of all life forms and the relatedness of all living beings. A prominent characteristic of both prokaryotic groups, Bacteria and Archaea, is their remarkable chemical diversity in terms of energy metabolism (Figure 1.12): Although these cells retain a relatively simple morphology, and a small size, which is optimally suited for interaction with their environment, they have evolved the ability to utilize a Figure 1.12. Unity and diversity of life. While bacteria and archaea exhibit wide range of substrates and extraordinary biochemical diversity with respect to energy metabolism, eukarya energy sources. They can evolved morphological diversity as multicellular organisms. use a vast array of different electron donors, and as a result can form many different products, making prokaryotes highly versatile in terms of their energy metabolism. On the other hand, the Eukarya domain features a higher level of cellular complexity than prokaryotes (Box 1.3), and their cellular compartmentalization requires more regulatory checkpoints for cellular tasks compared to prokaryotes. Major eukaryotic groups have evolved ways to achieve tight cooperation between cells, a prerequisite for the formation of multicellular organisms, enabling cells to specialize in function, as a sort of "division of labor". This specialization has led to a wide range of morphological diversity, both at the cellular and 15 at the organismic level, which is evident in the wide range of forms and associated functions of eukaryotic organisms and organs. Historically, large multicellular eukaryotes were the focus of scientific attention due to their size, which made them more readily observable and amenable to description. Their study led to the establishment of the classical fields of biology, namely zoology and botany. However, with the invention of the microscope in the 17th century (Figure 1.13), scientists such as Antonie van Leeuwenhoek and Robert Hooke began to discover small unicellular life forms, which today are the subject of the field of microbiology. Later, towards the end of the 19th century, other scientists such as Robert Koch, Louis Pasteur, and Martinus Beijerinck developed techniques to culture and isolate different strains of microorganisms. For a long time, prokaryotes were considered as a single group of organisms. However, the advent of Figure 1.13. The first microscopes revealed molecular methods enabled researchers to microscopic life. (A) Microscope built by Antonie van compare actual genetic sequences and the Leeuwenhoek (1632-1722) (10 cm large); (B), detailed structure of cellular components, Microscope by Robert Hooke (1635-1703); (C). Drawings by Robert Koch and Ferdinand Cohn from 1876. Image revealing that prokaryotes in fact are credits: (A) Alamy ID FFA98D, (B) Alamy HRP69, (C) composed of two distinct groups or domains of Farlow Botanical Library, Harvard University. life: Bacteria and Archaea. Box 1.3. Cells: the central units of biology (1-5 µm) (10-100 µm) Figure 1, Box 1.3. Comparison of typical prokaryotic (bacterial and archaeal) and eukaryotic cells. All cells contain genetic information, either in the cytoplasm (nucleoid) of prokaryotic cells or within the nucleus of eukaryotic cells. Eukaryotic cells are more complex, containing a nucleus with chromosomal DNA as well as mitochondria and other organelles. 16 Cells are the fundamental units of life capable of self-reproduction. They function as compartments, separating the internal cytoplasm from the external environment. Prokaryotic cells, typically measuring 1 to 2 µm, are much smaller than eukaryotic cells (Figure 1 Box. 1.3), which usually range from 10 to 100 µm in diameter. Eukaryotic cells possess a nucleus and various other internal compartments, such as mitochondria, which originated from free- living bacteria. For now, we will focus on simpler prokaryotic cells, which evolved earlier and dominate in terms of numbers on Earth. The conventional view of a prokaryotic cell as a membranous bubble enclosing a reaction space with DNA and some proteins floating in a watery cytoplasm is misleading. In reality, the interior of a cell is densely packed (Figure 2 Box 1.3), with biological macromolecules competing for space in a crowded molecular environment much like a real estate market. Figure 2, Box 1.3. Molecular contents of an E. coli bacteria cell in absolute numbers. The illustration on the left shows the crowded cytoplasm of a bacterial cell (image courtesy of D. Goodsell). The cartoon on the right shows approximate numbers of different types of molecules in E. coli, rounded to an order of magnitude. Figure taken from Phillips et al., 2013 "Physical Biology of the Cell", 2nd edition, Garland Science. The DNA content of the best-studied bacterium Escherichia coli (E. coli) amounts to 4.6 million base pairs on average, but genome sizes in E. coli strains may vary by 20% or more among strains due to horizontal gene transfer and gene loss. A typical bacterial gene is about 1000 base pairs long, resulting in around 4,300 protein-coding genes in a standard E. coli cell. These genes are expressed to varying extents at different times, reflecting the highly dynamic nature of the proteome, which is the sum of all proteins at any given moment. This plasticity is the result of the cell’s continuous response to internal signals and environmental cues. In fact, under well-defined laboratory conditions, only about 500 genes are required for the minimal functional requirements of an E. coli cell. While DNA only accounts for 3% of a bacterial cell’s dry weight, RNA molecules account for approximately 20% of the cell’s dry weight, predominantly as components of ribosomes, the molecular structures that translate mRNAs into polypeptide chains as we will discuss in Chapter 4. The approximately three million protein molecules in the cell contribute more than half of the dry weight (55%) (Figure 3 Box 1.3). Of these proteins, about two-thirds reside in 17 the cytoplasm, while the remaining one-third is embedded in the cell membrane. The cell membrane of E. coli is composed of more than 20 million phospholipid molecules, representing approximately 9% of the cell’s dry weight. These diverse molecules are endowed with unique properties and functions, including catalyzing reactions, serving as structural elements, and separating the interior from the exterior, aspects that will be further explored in Chapter 3. Figure 3, Box 1.3. Relative composition of an E. coli bacterial cell. The graphical representation shows the relative fraction of different constituents in the dry mass of a cell. Data are based on average E. coli cells growing at 37 °C under defined conditions on glucose in the presence of molecular oxygen (aerobic conditions). Cell dry weight data are redrawn from Milo & Phillips, 2015, "Cell Biology by the Numbers", Taylors and Francis and C. Neidhardt et al., "Physiology of the bacterial cell", Sinauer, 1990. "Trees of Life" The impulse to classify living beings is perhaps as old as mankind itself. In 1735, Carl Linnaeus, in an attempt to describe the divine genesis of all life forms, developed the first classification system based on morphological similarities between organisms. In his catalogue of living beings, 'Systema naturae', Linnaeus grouped organisms in nested hierarchies with two kingdoms, plants and animals, at the top. Linnaeus also formalized the binomial nomenclature, which is still used today to name a species. However, Linnaeus did not interpret the organisms he classified in an evolutionary context. Consistent with the prevailing beliefs of his time, Linnaeus regarded living beings created according to a plan, fixed and unchanging. This view began to crumble as the result of several observations. Georges Cuvier was one of the first scientists to systematically study fossils, biological material that turned into stone, and he is considered one of the founders of the science of paleontology. Cuvier realized that fossils represent the remains of extinct species and developed a system to classify fossils based on their distinct characteristics and comparative anatomy from which he proposed a succession 18 of different forms of life through time. Another important line of thinking refers to geology. It was Charles Lyell who proposed the principle of uniformitarianism: the idea that the same natural processes that shape Earth today have been operating throughout its history. Lyell's principle suggests that the sediments present today are the result of a gradual accumulation of sediment and can be used to understand how they formed and transformed over billions of years. He famously stated "The present is key to the past" in his book Principles of Geology, which was first published in 1830. His ideas were influential not only in geology, but also in biology, as they paved the way for the understanding that living beings, too, are subject to constant change. Figure 1.14. Charles Darwin and Alfred R. Wallace. In 1858, Darwin and Wallace independently published treatises on the theory of evolution by natural selection. Darwin’s book "On the Origin of Species by Means of Natural Selection" was published in 1859. Image credits: portrait C. Darwin, Huntington Library; “Tree of Life”, the first-known sketch by Darwin of an evolutionary tree describing the relationships among groups of organisms; Syndics of Cambridge University Library, portrait AR Wallace: Getty Images, Hulton Archive. This revolutionary thought was brought forward independently by Alfred Wallace and Charles Darwin (Figure 1.14), even though later Darwin’s book On the Origin of Species by Means of Natural Selection (published in 1859) became much more widely known than Wallace’s essay. Darwin presented evidence that the diversity of life arose through "descent with modification" from a common ancestor. Thus, the study of present-day organisms allowed inferences about past forms (Figure 1.15). As individuals compete for limited resources in the so-called "struggle for life", those individuals that are more suited to the environment are more likely to survive and reproduce. Their heritable traits therefore have a greater chance of being passed on to future generations than those of individuals less adapted to the environment, who are less likely to survive and reproduce. As a consequence, the process of natural selection leads to the gradual evolution of populations over time. 19  Figure 1.15. Evolution by natural selection. (A) ‘Darwin's finches’, collected during the Beagle voyage to the Galápagos islands, played a crucial role in developing the theory of evolution by natural selection. In 1939, Darwin wrote that "It is very remarkable that a nearly perfect gradation of structure in this one group can be traced in the form of the beak (…)". (B) Darwin’s scheme of gradual change through time. (C) Key points of Darwin’s and Wallace’s theory: gradual descent with modification from a common ancestor and natural selection. Relationships between organisms can be inferred from extant organisms, as done by Darwin and Wallace. (D-E) Experimental evolution of E. coli on solid growth medium containing a gradient of antibiotics, conducted by Roy Koshony and colleagues. The images monitor the emergence of lineages over 12 days. As E. coli grows, random mutations occur. Only individuals with mutations that confer resistance to a higher concentration of antibiotics are able to grow at higher antibiotic concentrations and give rise to new lineages. Lines in (E) indicate video-imputed ancestry. The white frame indicates the region that is shown at different time points in (D). Images from Baym et al., 2016 Science 353:1147-1151. Darwin, of course, had no understanding of how evolutionary principles acted at the molecular level, since DNA was only discovered much later to encode hereditary information. From today’s perspective, however, the modifications in heritable traits described by Darwin originate from random mutations in genetic sequences. These mutations occur by chance and are the source of genetic variation on which natural selection acts. Mutations are neither inherently good or bad, but depending on the environment, they may confer an advantage or disadvantage to their carrier or may be simply neutral. Prokaryotes evolve not only by mutation, but also by acquisition of new genes from other lineages whereby gene acquisition (or loss) can be seen as a kind of mutation. In the process of natural selection, mutations might provide organisms with a selective advantage allowing them to occupy a new niche or outcompete others, thus benefiting from the innovation (Figure 1.6). The traits that confer these advantages are passed on through generations, and additional mutations can occur, leading eventually to entirely new lineages of organisms. Today, evolution is directly observable in real-time laboratory experiments. For example, Figure 1.15 D-E shows the gradual development of resistance towards an antibiotic by the bacterium Escherichia coli. This empirical approach allows researchers to follow evolutionary dynamics live and also enables mapping of the sequence of genetic changes (genotype) that confer a particular property (phenotype). Unfortunately, if we want to infer the evolutionary 20 history and the relationships between living species, we cannot go back in time. A reverse approach is therefore needed to look retrospectively into the past. This is the aim and scope of phylogenetic classification systems. Over the last century, numerous phylogenetic systems, or "Trees of Life," have been proposed, documenting the concurrent evolution of all living organisms and establishing the conceptual frameworks governing their relationships. Ernst Haeckel introduced the notion of a monophyletic tree of life, positing that all life forms derive from a common ancestor, with their evolutionary relationships represented as separate branches – a concept famously illustrated in his 1866 "oak tree of life" (Figure 1.16). Nevertheless, Haeckel’s tree and subsequent models were largely based on a limited number of morphological characteristics. This can lead to errors in the designation of branching points, as traits can be mistakenly thought to be monophyletic (from a single common ancestor) when they are actually polyphyletic (arising independently in different lineages). A pertinent example is the ability to fly, which we now know evolved independently in birds, bats, and insects, contrary to earlier assumptions. Figure 1.16. Tree of Life drawn by Ernst Haeckel. Based on Darwin’s and Wallace’s theories, Haeckel developed the first phylogenetic tree of life (1866). The tree, which is also known as an oak tree, illustrates a monophyletic origin of life. In addition to showing plants and animals as two major divisions of life forms, he included protists, comprising all microscopic organisms known at the time. The group ‘monera’ at the base of the tree corresponds to what we now call bacteria and archaea. Haeckel concluded that all organisms are the descendants of such autogenous “Moneren”. (Source: Ernst Haeckel, Allgemeine Entwicklungsgeschichte der Organismen, Berlin 1866; Library University of Darmstadt, Germany). 21 But what kind of evidence can shed light on the largely unknown evolutionary paths from the past? The answer is provided by molecular data. Genetic information has been passed on from generation to generation. Occasionally mutations occur, some of which become fixed in populations over time. We can therefore conclude that two organisms sharing similar genetic material are closely related (with some caveats that will be discussed below). Conversely, organisms that are only distantly related will show increasingly large differences in their genetic sequences. Starting in the late 1970s, Carl Woese and his colleagues applied this logic and concentrated on ribosomal RNA (rRNA) sequences to reconstruct a molecular tree of life. Woese had a number of good reasons to choose rRNA as a phylogenetic marker. Ribosomes are responsible for the translation of messenger RNA (mRNA) into proteins, as we will discuss in detail in Chapter 4, and are universally conserved, performing exactly the same function in all cells. Because the process is complex, involving many intricately linked steps that must work as an ensemble in every ribosome and every cell, they must be highly conserved. This means that change only occurs slowly over time as most changes are penalized, resulting in non-viable offspring. However, despite the slow rate of change of the molecules that make up the ribosome as a whole, their genetic sequences contain enough variability to provide deep insights into evolutionary relationships. In 1990, Woese and his colleagues published a paper on the "natural systems of organisms", in which they proposed to split prokaryotes into two different domains, Archaea and Bacteria, resulting in three domains tree of life (Figure 1.17a). "Molecular comparisons show that life Figure 1.17. Phylogenetic tree based on marker genes. (A) A phylogenetic tree constructed using the sequences of ribosomal RNA genes shows the division of living organisms into three domains: Bacteria, Archaea and Eukarya (modified from Stetter 1996 FEMS Microbiol Rev 18:149-158 based on Woese et al. 1990 PNAS 87:4576-4579). Thick lines indicate hyperthermophilic organisms. Note that mitochondria and chloroplasts also contain ribosomal DNA which relates to their bacterial origin (not shown). (B) Unrooted phylogenetic trees based on 35 phylogenetic marker genes and different models of inference show two alternative evolutionary scenarios. Accurately positioning long branches in phylogenetic trees is challenging because they tend to group together regardless of their true relationships, an artifact called ‘long branch attraction’. Recent analyses suggest that Eukarya may have emerged from Archaea related to modern Asgard archaea (tree on the right) (modified from Williams et al., 2020, Nat Eco Evo 4:138-147). 22 on this planet divides into three primary groupings", they wrote and concluded that the new classification system recognizes "that, at least in evolutionary terms, plants and animals do not occupy a position of privileged importance". In the past three decades, genome sequences have been obtained from many cultivated and uncultivated bacteria and archaea, expanding the tree of life and leading to alternative scenarios for placing eukaryotes with or separate from archaea (Figure 1.17b). When scientists started to look at the sequences of other evolutionarily conserved genes, they realized that genomes are structured like a mosaic. In other words, each gene, which encodes a protein with a certain function, has its own history. When looking at all genes encoded in the genomes of present-day Eukarya, it was noted that some genes are more closely related to sequences from extant Archaea and other genes are clearly related to sequences found in extant Bacteria. The explanation for these findings is that Eukarya are chimeric, that is, derived from more than one organism. Several researchers, including Konstantin Mereschkowsky, postulated in 1910 that the chimeric nature of eukaryotes originated from symbiotic relationships, via a process they called symbiogenesis. Based on this work and on later discoveries showing that chloroplasts and mitochondria both contain their own DNA and have other prokaryotic features, Lynn Margulis refined the endosymbiotic origin of eukaryotes in 1967, the partnership of cells within other cells. This endosymbiotic theory today contends that (1) mitochondria descend from bacteria that were stably incorporated into an Archaeon, and that (2) chloroplasts are the result of the incorporation of a photosynthetic cyanobacterium by a eukaryotic cell. Sequencing data suggest a bacterium most closely related to extant alphaproteobacteria as the origin of mitochondria and a member of an archaeal lineage (Asgard archaea) as the closest relative of the original host cell (Figure 17b). Considering the ribosomal tree and other markers for information processing in the cytosol, Eukaryotes emerged from within the domain of Archaea, but if we consider mitochondrial ribosomes, they emerged from the bacteria. Eukaryotes simultaneously sit on both the bacterial (mitochondria, energy conversion) and the archaeal (cytosolic, information processing) branches in the tree of life. They are the product of endosymbiosis. These endosymbiotic events imply that the tree of life can also be drawn in a different way (Figure 1.18). Around 4 billion years ago, the first prokaryotic cells emerged from geochemical and prebiochemical processes. LUCA, the last universal common ancestor, gave rise to both Bacteria and Archaea, which were the only domains of living beings on Earth for more than a billion years, as discussed above. Then, however, in a unique process, bacterial cells were acquired by an archaeal cell or group of cells, marking the start of the eukaryotic common ancestor and eventually LECA, the last eukaryotic common ancestor from which all Eukaryotes originated. From then on eukaryotes existed and evolved in parallel with bacteria and archaea, often in symbiotic relationships, especially between eukaryotes and bacteria. A second endosymbiotic event involving a cyanobacterium and a eukaryote gave rise to chloroplasts and the founders of the plant lineage. Endosymbiosis explains the chimeric nature of eukaryotes, with different genes most closely related to their counterparts in either bacteria or archaea (Figure 1.18). It can be considered 23 a tremendous accelerator of cellular change in the evolution of life because it combines the capabilities of two cells as a unit. As mentioned above, after the emergence of mitochondria, another endosymbiotic event occurred, resulting in the formation of chloroplasts in a branch of Eukarya, leading to the emergence of algae and plants. During the life cycle of eukaryotes, meiosis and sexual reproduction emerged, when the genomes of two organisms from the same species are mixed in one cell and redistributed to offspring. Figure 1.18. Modern view of the "Tree of Life". Bacteria and Archaea (prokaryotes) descended from the last universal common ancestor (LUCA). Eukaryogenesis and the origin of the last eukaryotic common ancestor (LECA) involved the endosymbiosis of a bacterial endosymbiont, which became the membrane-bound mitochondrial organelles of eukaryotes, in an archaeon host. Eukaryotes are categorized into supergroups, with algae and plants arising from secondary endosymbiosis with cyanobacteria. Modified from Martin et al., 2017, MMBR 81:e00008-17. The exchange of genomic information is also fundamental for prokaryotes, and probably has been since the earliest stages of life. The genomes of extant organisms attest to a chimeric nature resulting from the integration of foreign DNA into genomes. This process, known as horizontal gene transfer (HGT), allows the exchange of genetic material even between distinct species, profoundly shaping evolutionary trajectories. In this process, pieces of DNA are transferred through various mechanisms including infection by viruses or by uptake of environmental DNA, providing prokaryotes access to a wider range of functions than those obtained from their respective parent cell. To illustrate such transfer, consider the rapid spread of antibiotic resistance in bacteria and the consequences for treating pathogenic bacteria in hospitals. This spread is due to the presence of genes for antibiotic resistance located on plasmids, circular extrachromosomal DNA molecules that can be readily transferred among pathogens. 24 Key points: ▪ Earth provided the elements and conditions for life to emerge, and life in turn has had profound effects on Earth ▪ Bacteria and archaea were the only known organisms until halfway through Earth’s history when eukaryotes emerged ▪ Sequence information for ribosomal RNA is commonly used to infer the relatedness of all organisms to each other ▪ Mutations cause changes in genomes. Bacteria and archaea can exchange genetic material independent of reproduction, while eukaryotic sexual reproduction involves combining entire genomes from two different but related organisms Questions 1) Compare the physical and chemical conditions on Earth at the time when life first emerged with today's conditions. Find at least two reasons why animals could not have existed on Earth at that time. 2) What do some scientists mean by saying that the ribosomal tree is the "tree of 1%"? Origin of life The biology of organisms changes over time. It evolves as a consequence of natural selection and neutral change. The composition of organisms and their biomass on Earth today (Figure 1.3) is the outcome of billions of years of evolution of life on our planet (Figure 1.11). Let us now look back in time and discuss some of the ideas that have been put forward to explain how life might have originated. The topic is inherently difficult from a scientific perspective due to the lack of direct historical evidence and experimental limitations. Curiosity about life’s origin is fundamental and old. From the time of Aristotle and the ancient Greeks until the 17th century, it was generally believed that life arose from nonliving matter via spontaneous generation. But in 1668, Francesco Redi put spontaneous generation to the test by placing pieces of meat in either open or sealed containers. Redi observed that flies could deposit their eggs in the open jars and maggots would form in the rotting meat within a few days. However, no maggots appeared in the sealed jars. Redi concluded that maggots could not spontaneously emerge from rotting meat. He summarized his findings in the famous phrase omne vivum ex vivo, which in Latin means "all life [is] from life." Because of the inclusion of a control condition in this simple setup, Redi is considered as one of the founders of experimental biology. 25 Almost 200 years later, Louis Pasteur conducted what is known as the swan-neck bottle experiment to test whether simpler life forms, microorganisms, arise spontaneously or from preexisting cells by division. His experimental equipment consisted of bottles with long curved tubes that allowed free exchange of air but trapped dust and air-borne microbes in the bend. After filling the bottles with meat broth and sterilizing the liquid by heating, Pasteur saw that the liquid remained sterile – and could only be populated by microorganisms if the bottles were tipped and the liquid came in contact with the microbes trapped in the bend. Today, the process of sterilizing materials is called pasteurization in his honor. From his experiment, Pasteur concluded that living organisms only arise from parent organisms similar to themselves. Although this principle is still pertinent today, it leaves one big question unanswered: How did life start in the first place? Specifically, how did inanimate matter evolve into complex systems capable of utilizing energy from its environment for growth and self-replication? Essential conditions To explain the origin of life, numerous hypotheses have been put forward and are hotly debated. These differ with respect to the environment and environmental inputs that they assume to enable the emergence of life. There is currently no consensus. There is also no consensus as to whether the emergence of cellular life is deterministic or even predictable (at least in principle) or whether it was the result of chance, the outcome of many random events. However, there is some agreement about the general requirements that had to be met for the transition from chemical reactions on the early Earth to the first cellular life (Figure 1.7), among them: ▪ Some sort of inorganic catalysts promoted the abiotic synthesis of small organic molecules. Chemical reactions by such inorganic catalysts open up the possibility of self-organizing networks of chemical reactions that operate as autocatalytic cycles. ▪ Reactive forms of C, H, N, O, P and S were available and there must have been a stable source of energy that enabled evolving, pre-biotic systems to polymerize small building blocks into larger macromolecules and to maintain a state far from thermodynamic equilibrium. Life always obeys the laws of thermodynamics and must involve the release of free energy. ▪ Barriers existed that permitted physical compartmentalization, a separation of an inside from an outside. Gradients are essential to life. On the inside, organic molecules, including polymers, must have accumulated to high concentrations, so that pre-biotic metabolic networks could form. At the same time, by-products of these networks must have been continuously synthesized on the inside and removed to the outside. This barrier should not seal off the interior completely from its environment, as matter also needs to enter from the outside in order to supply the substrates for chemical reactions. There is considerable ongoing debate about these fundamental aspects, the specific environments, and the sequence of events that led to the formation of protocells. Some scientists argue that metabolism came first, with inorganic catalysts playing a key role in 26 establishing autocatalytic metabolic networks in the beginning. Others place greater emphasis on molecular replication and hereditary molecules, focusing on conditions that would be favorable for the synthesis of nucleic acids. The problem is that we do not know what the actual intermediate stages in the transition from non-life to life were. Researchers are trying to make protocells, but we do not know what real protocells looked like, because even the simplest known unicellular organisms known today are highly complex systems. Extant bacteria and archaea present all the various features (metabolism, membranes, hereditary molecules, the genetic code) that characterize every living entity on Earth. As yet, life has also not been created "ab initio" under laboratory conditions and even if it were possible, it still would not prove that life emerged on Earth along a similar path some 4 billion years ago. There are two different general approaches to address the origin of life problem (Figure 1.19a), top-down and bottom-up. The bottom-up (or synthetic) approach involves studying plausible planetary environments, chemical reactions and geochemical processes that could have contributed to the formation of complex molecules from simpler starting materials. The top- down (or inferential) approach involves comparing today’s unity and diversity of organisms to Figure 1.19. Origins of life scenarios. (A) Complementary approaches to inferring the features of the last universal common ancestor (LUCA). "Biology down" employs knowledge about phylogenetics and the biochemistry of known organisms, while "Geochemistry up" tests prebiotic chemistry conditions based on plausible early life environments, to generate hypotheses about the origins of life. (B) Deep sea hydrothermal vents (white smokers) (figure courtesy: NN). These vents were originally discovered by Kelley et al. 2001 Nature 412:145-149. (C) Vent deposits from Lost City with porous structures that allow the mixing of seawater and hydrothermal fluids within the interior walls. Image courtesy of Kelley et al., 2005, Science 307, 1428-1434 (D) The redox, pH, and temperature gradients at submarine hydrothermal vents have been proposed as sites for the origin of life by W. F. Martin and M. Russell. This hypothesis suggests that iron monosulfide compartments within these vents could have served as membrane-bounded prokaryotic cells with DNA, RNA, ribosomes, and proteins before the emergence of distinct membrane types found in bacteria and archaea. This scheme is redrawn from Martin and Russell, 2003, Phil. Trans. R. Soc. Lond. B 358:59-85. 27 identify ancient traits from derived traits. This information can then be used to generate inferences about life’s beginnings and to extrapolate the properties and features of LUCA, the last universal common ancestor. Ideally, both approaches would converge in a common view on the origin of life. Some scenarios are presented below. They are intended to illustrate how difficult this problem is and to highlight some of the considerations put forward, without being exhaustive. Scenarios for the transition from geochemistry to biochemistry Although Darwin did not write about the beginning of life in his book 'On the Origin of Species', he later speculated in a letter to a friend that life may have started in a "warm little pond". In the 1920s, independently of each other, Alexander I. Oparin and John B.S. Haldane speculated that life on Earth arose through a progressive chemical evolutionary process. At the time, the molecular basis of cellular biochemistry was still largely unknown, including the nature of heritable information. Accordingy, their scientific ideas about origins were general, not specific. They reasoned that inorganic compounds (such as carbon dioxide (CO2), ammonia, and water vapor) contained in the anoxic atmosphere of the early Earth reacted with each other under ultraviolet light and, by a slow process of molecular evolution, gave rise to more and more complex organic molecules that accumulated over time in the primordial oceans. The idea of a "primordial soup" gained traction in 1953 when Stanley Miller and Harold Urey demonstrated that inorganic precursors could be transformed into organic molecules such as amino acids and other basic building blocks of major biochemical polymers using electric discharges that simulated natural lightning as the source of energy to drive the reactions. The significance of their experiment was to show that essential molecules of life can arise from simple chemical reactions under putative early Earth conditions. Planetary scientists now believe that the Earth’s atmosphere was less reducing than assumed by Miller and Urey and contained a different mixture of gases, mostly molecular nitrogen (N2) and carbon dioxide (CO2), rather than the high concentration of ammonia (NH3) that was used in the "Miller-Urey" experiment. Even if lightning could have provided enough energy to synthesize simple organic molecules, it would not have been a sufficiently stable source of energy to maintain pre-biotic systems far from thermodynamic equilibrium. Apart from processes resulting in building blocks driven by lightning, chemists have proposed alternative synthetic routes using particularly reactive molecules such as cyanide to produce nucleotides for example. Once prebiotic chemistry had produced such organic molecules, polymers must have formed. The concept of RNA as a primordial molecule was hypothesized by Francis Crick, Leslie Orgel and Carl Woese. The hypothetical stage in the evolutionary history of life on Earth, in which self-replicating RNA molecules proliferated before the evolution of DNA and proteins, is also referred to as the 'RNA world', a term coined by Walter Gilbert. RNA molecules could store information and catalyze chemical reactions, including their own synthesis. In fact, Manfred Eigen and colleagues showed in the 1970s that an RNA molecule can template the exponential synthesis of new RNA molecules. RNA is still catalytically active in all cells, in ribosomes, as we will see in Chapter 4. According to the RNA world hypothesis, RNA is believed to have preceded more complex biological systems that 28 rely on proteins or peptides for catalysis and on DNA for information storage. The hypothetical stage is also referred to as 'RNP world' (RNP for ribonucleoprotein), as proposed by Thomas Cech. Complex, self-replicating RNA molecules may have provided the basis for the transition to peptide synthesis directly on (non-canonical) RNA, from which ribosomal peptide synthesis may have evolved. According to this line of thought, an early co-evolution of RNAs and peptides could then have given rise to a nucleic acid-protein world, and the generation of proteins as the predominant catalysts in cells today. Independent scenarios on the origin of life place emphasis on abiotic catalysts and metabolism at the origin of life. These theories are tied to the geochemistry of deep-sea hydrothermal vents, which were discovered near the Galapagos Islands in 1977. These "black smokers" arise at submarine fissures that form at spreading zones, places where tectonic plates are moving apart from each other as magma emerges from the Earth’s mantle. They discharge superheated (400°C) water that is rich in dissolved minerals from the Earth’s crust. These minerals precipitate as insoluble sulfides when the hydrothermal effluent comes in contact with cold ocean water, forming black clouds and impressive mineral-rich chimney-like mounds (Figure 1.19). The discovery of black smokers stimulated John Baross in the 1980s to propose that life originated on the ocean floor. He argued that mineral catalysts generally preceded enzymes in evolution, potentially at hydrothermal vents. In the 1990s, Günther Wächtershäuser further developed those ideas into what he called an "iron-sulfur world" that envisaged inorganic gases reacting on the surface of iron-sulfur minerals, producing organic compounds and releasing small amounts of free energy that could promote further biochemical reactions. In this scenario, purely geochemical reactions set the stage for life to emerge at the surfaces of these chimney-like structures. However, there is also an important caveat. The water coming out of the black smokers reaches temperatures over 300°C. Large organic polymers would not be stable at this extreme heat. Theories on the origin of life at the ocean floor gained new momentum when Deborah Kelly and colleagues discovered Lost City, a hydrothermal field at the mid-Atlantic ridge, in 2000. In contrast to black smokers, these hydrothermal vents emit white clouds (Figure 1.19) and release alkaline water at "only" 70°C. The alkalinity of the hydrothermal fluid is a result of a natural chemical process called serpentinization, whereby sea water reacts with the mineral olivine contained in the Earth’s crust, leading to the formation of serpentine and hydrogen (H2). These alkaline vents contain microscopic compartments, like fossil structures discovered by geologist Michael Russel, who had proposed that iron sulfide bubbles containing alkaline hydrothermal solutions could have served as cradles of life (Figure 1.19). As pointed out by William F. Martin and colleagues, such structures are interesting in this context for several reasons: They have the capacity to concentrate molecules rather than losing them to the open ocean. The liquid in alkaline vents contains minerals that are strikingly similar to (Fe, Ni) S centers in enzymes of extant and early diverging lineages of prokaryotes, specifically methanogenic archaea and acetogenic bacteria. These organisms live off H2 and CO2 and harness their conversion for energy and biomass needs as anaerobic organisms that live in the absence of molecular oxygen. Thus, biological processes in present-day organisms resemble the geochemistry found at deep-sea hydrothermal vents such as Lost city. In the oceans 4 billion years ago, there would have been a steep pH gradient across the walls of such compartments as the atmosphere at the time contained considerable amounts of CO 2 29 that was partially dissolved in the oceans, acidifying them, and the alkaline effluent, with a pH of 9 or higher, produced at the vents. This pH gradient may have constituted a natural proton motive force, which we will discuss again in Chapter 6, already in place, a consequence of an acidic ocean interfacing with a hot, alkaline fluid through a precipitated abiotic membrane: the same force that today drives the synthesis of ATP, the universal energy currency used by all living organisms. The convergence between the geochemistry (bottom-up) and the biochemistry of life (top- down) goes further when considering methanogens and acetogens. By comparing the sequences of more than 6 million protein-coding genes and grouping them into phylogenetic trees, William F. Martin and colleagues found about 350 protein families that they traced back to LUCA using a range of simple criteria. According to them, the inferred ancestor of bacteria and archaea was anaerobic, possessed ribosomes and used the genetic code to translate genetic information into proteins. It was replete with FeS clusters, used molecular hydrogen (H2) as an energy source and was capable of CO2 fixation. According to that analysis, LUCA was not a free-living cell, rather it was confined to a system of inorganic compartments. The similarities between the reactions catalyzed by minerals deposited at H 2-producing hydrothermal vents and ancient biochemical pathways suggest that the origin of life might have been linked to an almost seamless transition from geochemistry to biochemistry. Clearly, the mystery of life’s origin persists, and it always will, surrounded by alternative ideas for processes and environmental conditions. As Albert Eschenmoser once remarked, "The origin of life cannot be discovered; it needs to be reinvented". Reflective analysis and experimental testing of hypotheses about primordial processes will continue to motivate scientists to develop plausible scenarios that might elucidate the emergence of life. The topic is pivotal to the field of biology and is destined be the subject of research and discussion for many years to come. Key points: ▪ Interdisciplinary thinking and approaches are needed to develop plausible scenarios for the beginning of cellular life ▪ There is currently no consensus on the origin of life, but various hypotheses have been proposed to explain the path from geochemistry to biology Questions 1) Why were autocatalytic cycles crucial for the emergence of life? 2) Could new life forms emerge on contemporary Earth? 30