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: An 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...

Department of Biology Fundamentals in Biology 1: From Molecules to the Biochemistry of the Cell 1 Chapter 1: An 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): Current estimates for the number of living species range from 2 to 100 million or even 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 in some forests in North America that can grow up to 80 metres high. Nonetheless, all living forms share an important number of fundamental traits and, to the best of our knowledge, descend from a common origin. As such, and from an evolutionary perspective, all organisms are related to each other. Moreover, no life form exists entirely on its own: Every species and every single organism is interconnected and depends in one way or another on interactions with both abiotic (non-living) and biotic (living) factors in the environment. All of these connections and interactions 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. A selection of life forms. Shown are a praying mantia (top left), a fungus called witches butter (Tremella mesenterica) (top middle), a white rhinoceros, Diceros simus (top right), Rhinoceros beetle (Eupatorus gracilicornis) (bottom left), a diatome algae (bottom middle), giant sequoia trees (bottom right). 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 bacteria. But there are also direct, short- and long-term interactions. An interaction between organisms can be beneficial to one or more of the partners (positive interaction), harmful to one or more of the organisms involved (negative interaction), or have no substantial effect (neutral interaction). All of these interactions are summed under the term symbiosis, from the Greek word for "living together", which refers to any kind of interaction between different organisms. Such symbiosis 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 of organisms, different terms are used to describe various types of relationships among them 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 humans. They can transmit bacteria that cause Lyme disease and harm the host. Conversely, mutualistic relationships provide benefits to both partners. An ancient example of mutualism is the partnership of land plants and certain fungi in the soil, the mycorrhiza. Plants and mycorrhizal fungi evolved concurrently probably since the time when plants first colonized land (around 400 million years ago) and the relationship persists in most extant plant taxa. Figure 2. Examples of mutualistic symbiosis. Nodules of soybean plants (top left), lichens (top right), corals (bottom left) and a bee plant symbiotic relationship (bottom right). In this mutualistic interaction, plants supply their fungal partners with photosynthetic products, while the fungi help the plants obtain nutrients, in particular phosphorus, from the soil. Other examples of mutualistic relationships are those between nitrogen-fixing bacteria and leguminous plants such as soybean or pea plants, lichens that arise as a composite from fungi and algae or Cyanobacteria, corals that are animals that associate with unicellular phototrophs and the relationship between pollinators and flowering plants (Figure 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 about 10,000,000,000,000 (10 trillion) microbial cells produce vitamins and provide nutrients by helping to digest 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: Microorganisms 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. 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 lifeforms like plants and animals. 3 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 incredible amount of samples obtained using techniques including satellite-based remote sensing, counting by eye or using microscopes, to 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 the water content. They concluded that the sum of 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 3). The second largest group in terms of biomass is bacteria, which account for about 13% of the total sequestered carbon. All other taxonomic groups together, which are archaea, fungi, protists, and animals (including humans), add up to about 5% of the global biomass. Figure 3. Composition of the living world: Global biomass distribution by taxonomic groups. Each polygon area represents the relative fraction of the total estimated biomass (carbon) on Earth. Total biomass (carbon) on Earth ~ 550 Gt. Modified after 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 C reveals that humans and livestock such as cattle and pigs far outweigh the biomass of all wild mammals at present. Humans appeared just over 200'000 years ago and have contributed to a five-fold reduction in the biomass of wild animals in the last fraction of that time. These changes have been dramatic and rapid: If the entire Earth’s history were compressed into 24 hours, this reduction in biomass would have occurred only in the last second. While abrupt changes occurred in the past on Earth, think about the extinction of the dinosaurs, it is the first time that one species (humans) changed the Earth in such short time. Apart from the distribution of the biomass on Earth, the estimated number of living organisms in different taxonomic groups can be correlated. Bacteria and archaea were the first surviving organisms to have emerged on Earth probably almost 4 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 4 be, in the age of microorganisms. In terms of the number of organisms living today, bacteria alone comprise an estimated 1030 individuals, making them the most numerous. 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 4) shows that, in general, groups with a higher number of individuals also have a higher biomass. However, there is an exception to this rule: plants. Their abundance is smaller compared to the biomass 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 of six thousand tons. Figure 4. Relationship between abundance and biomass: In general, taxonomic groups with a higher number of individuals have a higher biomass. Plants are an exception due to the high biomass that many plant individuals can accumulate. Source: Bar-On et al. 2018 PNAS 115:6506-6511. 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 the majority of 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 5). Back to the overall distribution of biomass on Earth, now not by major groups of organisms, but by ecosystems at large. Unchallenged at the top, terrestrial ecosystems host the largest share of the Earth’s total biomass (Figure 5). In contrast, marine biomass is almost a hundred times smaller, even though 71% of the Earth’s surface is covered by oceans. Despite the large difference in biomass on land and in the oceans, the primary productivity, or the amount of carbon captured from carbon dioxide (CO2) mostly by photosynthetically active organisms per 5 Figure 5. Biomass distribution across different environments on Earth (on the left). Plants and fungi live predominantly in terrestrial systems, protists and animals predominantly in the oceans and bacteria and archaea are predominantly found in deep subsurface systems (on the right). Modified after Bar-On et al. 2018 PNAS 115:6506-6511). Figure 6. Distribution of biomass between producers (autotrophs) and consumers (heterotrophs without deep subsurface) in the terrestrial and marine environments. The size of the bars corresponds to the quantity of biomass of each trophic mode. Numbers are in gigatons of carbon. Modified after Bar-On et al. 2018 PNAS 115:6506-6511). year, is roughly equal in both environments. As a result, the CO2 conversion per biomass is different on land compared to that in the oceans, where the carbon turnover is much higher. In addition, and related to this aspect, the ratio of primary producers (the so-called autotrophs that fix CO2) and consumers (heterotrophs that take up organic carbon) is also different in the two environments. On land, the ratio of primary producers to consumers who feed directly or indirectly on the former is about 20:1, whereas in the oceans, the ratio of primary producers to consumers is inversed with 1:5 (Figure 6). Biology = Physics + Chemistry + Evolution This simple equation is not meant to be a mathematical formula, but rather to express the idea that biology, the study of living organisms, is based on principles from physics, chemistry, and evolution. Physics and chemistry provide a fundamental understanding of the processes that occur within living organisms. The laws of nature govern the universe, and therefore all living beings must abide by them. Physics as the most fundamental discipline, informs biology with its mathematical descriptions of natural phenomena, such as 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 the principal rules of physics and chemistry, biology is defined by evolution. Evolution explains gradual changes that occur over time to produce the diversity of life 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." Together, physics, chemistry, and evolution form the foundation of biology and are essential for understanding how living organisms function and interact with their environment. It is no coincidence that the chemistry of life is based on carbon (C), a highly abundant element on Earth that can form long chains that are both stable and flexible, as we will see in Chapter 2. Carbon is the central element of organic chemistry because it enables the generation of diverse molecular structures exploited for the emergence of life. Throughout the course of 6 life's history, organisms have increased their capacity to produce complex organic molecules. All major cellular compounds contain carbon, and in such large amounts that it alone accounts for half of the biomass dry weight on Earth, serving also as good proxy for biomass, as shown in Figure 3. According to the second law of thermodynamics, 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 visibly maintain themselves in a highly ordered state, therefore seeming to contradict the second law of thermodynamics. Likewise, while exergonic reactions (i.e., chemical reactions with a net release of free energy) move in the direction of greater probability, endergonic reactions (such as those required for the build-up of proteins or the polymerization of DNA) must be forced in the opposite direction, i.e., toward greater improbability, by coupling to other reactions. This coupling of endergonic reactions to exergonic reactions 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 do not exist in isolation. What is Life? It is anything but trivial to define life and there are many different ways of approaching the problem. In 1944, the physicist Erwin Schrödinger published a famous essay called "What is life?" in which he associated life with two aspects. First, life feeds on negative entropy or "negentropy". The term relates to the second law of thermodynamics that seems to be violated by life as we discussed above. According to Schrödinger, an organism maintains itself at a fairly high level of orderliness by continually "sucking orderliness from its environment". Second, Schrödinger emphasized the existence of hereditary material, which 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". Fusing these two rules together, Nick Lane, William Martin, and colleagues concluded 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 underpin an essential property of life: evolution of over time. Centuries-long discussions have not resolved the question about the features essential to life, and there is still no universally 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 matter to accumulate and interact with each other, but we cannot determine the exact point at which this prebiotic evolution crossed the minimal threshold of life (Figure 7). Life itself can be considered a major innovation, followed by major innovations in life’s history as will be discussed later in this chapter. 7 Figure 7. Simplified scheme to illustrate the increasing complexity in biology. Geochemical processes lead to the accumulation of organic molecules, which chemically interacted with each other and formed more complex systems crossing – approximately 4 billion years ago – the minimal threshold of "aliveness". Systems innovations became prevalent over time and were maintained, upon which other innovations are built on (illustrated by a line). Easier than defining life, it seems, is describing life and its key components, which relate to the characteristics we introduced above and to which we can relate with our current knowledge about the molecules and the biochemistry we have today. Most scientists agree with the view that an organism must fulfill four main criteria to be alive (Figure 8): First, an organism must have ways to convert and use energy to maintain itself, to grow and to divide. In other words, every organism needs some kind of fuel, which it will transform and metabolize before getting rid of waste products. As we will later see, energy is not just consumed or released during biochemical reactions, it can also be spatially stored and sustained in different forms such as Figure 8. Living beings fulfil three criteria: 1) All organisms have evolved ways to harness chemical energy from their environment to make copies of themselves. 2) Separate the inside of cells from the outside through. 3) All life forms pass information on to their offspring. 4) All living systems evolve according to the principles of Darwinian Evolution. Energy input (fuel) and nutrients, composed of elements, are required to build new cell biomass. Ultimately, cells proliferation is an autocatalytic process. 8 electrochemical gradients in cellular compartments, where separating membranes act as barriers. Membranes separate inside from outside, a second criterion critical to life. Living organisms must also have ways to pass on information to their offspring. Schrödinger’s code-script later turned out to be the sequence of bases in the chains of the nucleic acid DNA that makes up the genome. Cells as the fundamental unit of organisms replicate this genetic information before they divide and multiply. The information, the instruction to grow and copy is encoded in the hereditary material. It remains highly conserved to ensure "the copy of itself" as an autocatalytic process, but mutations, small changes in the genome, are always possible: given enough time and over generations, these alterations allow populations of organisms to adapt to changing conditions in their environment. These changes refer to a fourth criterion, all living systems have the ability to evolve. There is no life without random change followed by natural selection – the principles of Darwinian evolution. Figure 9. Illustration of a cell, the smallest unit of life with universal biochemistry. All life forms on Earth share common molecular features. These key components of life also guide the structure of this book, which focuses on core processes fundamental to every cell – and therefore deals with what could be called universal biochemistry (Figure 9). All life forms on Earth share common molecular features and rely on the same kinds of information-containing macromolecules, i.e., highly complex polymers such as DNA, RNA or proteins. These are in turn built from a common set of precursors within a wide chemical space as we will discuss in Chapter 2. 9 Key points: ▪ ▪ ▪ Organisms interact with each other in various ways, referred to as symbiosis. Plants dominate the biomass on Earth, while bacteria predominate in terms of number of living organisms. Life is characterized by the ability to harness energy, to assemble macromolecules from nutrients taken up from the environment, to 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 fridge 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 To understand the evolution of life on Earth, we need to look at the approximately 4.57 billion-year history of our planet (Box 1). It is important to note that interactions among organisms resulted in the diversity of organisms present to date (Figure 3) but there has been and still is also various twoFigure 10. Earth has provided a safe enough space for life to evolve, but at the same time life has also decisively shaped way interactions between the abiotic planet the actual geochemistry of our planet. These feedbacks are and life (Figure 10). While Earth provided thus both ways life on (surface) Earth and vice versa, resulting in a biosphere. Of note, the Earth is not just biologically alive, the fundamental conditions for life to arise, it is geologically alive, too. such as water and essential elements, life in turn has also greatly affected the actual geochemistry of the planet. One major example of such a life-on-Earth impact occurred about halfway through Earth's history, with the emergence of molecular oxygen (O2). This changed Earth's geochemistry in turn. Testimony of this life to Earth interaction is illustrated by O2 that reacted with soluble Figure 11. Banded Iron Formation (BIF). Red layers are Fe-rich minerals. They formed reduced iron (Fe2+), resulting in precipitation of iron as around 2.4 billion years ago. Their colour is due the presence of hematite (Fe2O3), a mineral 3+ Fe , which formed impressive iron layers in rocks to produced by the oxidation of ferrous iron (Figure 11), but also resulted in drastic during the erosion. transformations in the forms of life. Box 1. The planetary setting for the development of life In this box, we will cover the timescale of Earth and some of the requisites for life to emerge. The age of the Earth and how we know 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 occasionally occurs in rocks is zircon (ZrSiO4). Zircon contains the element zirconium (Zr) and forms in the magma of volcanoes. The mineral can then be dispersed as ash after the eruption and occasionally be trapped as volcanic layer when rocks form. Zircon, like many other 11 minerals, is not pure. The element uranium (U) can substitute for Zr in zircon due to its similar size. Uranium is interesting for dating the Earth: It decays to another element, lead, in a predictable manner and it does so very slowly. This decay is intrinsic to the element. In consequence, it allows researchers to determine how much time has passed since the rock was formed by measuring lead (Pb). Because the Earth has been and continues to be geologically active, with heat from its interior causing rocks to form and be destroyed, there are no remaining parts of the Earth that date back to its origin. Crucial for dating the Earth are some classes of meteorites that formed elsewhere in our Solar System, from the asteroid belt between Mars and Jupiter. These meteorites stem from bodies that cooled and solidified more quickly than the Earth, preserving their more ancient mineral makeup. Some meteoroids happen to fall on Earth and 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 part of biomolecules. Other elements, in particular carbon, nitrogen, phosphorous, and sulfur are also crucial for the existence of life. It is widely believed that stars and planets form – accrete – 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 volatile elements such as hydrogen, oxygen, carbon, nitrogen, phosphorous, and sulfur due to too low gravitational forces. Scientists concluded that these elements can be attributed to the collision of water-rich meteorites with Earth early in its history. 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 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 3) and the only planet where we know 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. Currently, human actions are having a substantial impact on the Earth, leading to a loss in biodiversity. The Sun is the primary source of energy that powers the surface of Earth, including life. Based on the Earth's distance from the Sun, we would expect the average temperature to be about 12 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 magmas. If CO2 were only being added to the atmosphere, it would build up and lead to a gradual increase in temperature. There are also processes that remove CO 2 from the atmosphere. One such process is chemical weathering, which occurs naturally, even before the existence of life on Earth, and continues to occur today. In this process, carbonate or biocarbonate, other forms of CO2, react with cations such as calcium (Ca2+) to form carbonate mineral, calcite, also known as limestone. The process of chemical weathering operates over vast geological timescales of thousands to millions of years. Another process that removes CO2 from the atmosphere involves life and is linked to carbon dioxide fixation (a process we will discuss in Chapter 7). However, only a tiny fraction of the organic carbon fixed by living organisms today is sequestered over long periods of time. This is in contrast to times far back in Earth's history, when plants first emerged and gave rise to what we now refer to as coal, a fossil fuel that derived from the compression of plant organic matter in terrestrial systems - or the remains of ancient marine plants and animals in marine systems, which led to the formation of oil, also known as petroleum. The proportion of CO2 in the total atmosphere in the preindustrial period was 0.00028, which means that for every million molecules of air, there are 280 molecules are CO2 (280 parts per million, ppm). This small amount of CO2 is crucial to the surface conditions of our planet. As of 2023, this amount has increased to 412 ppm leading to climate change. Relative to the total age of the Earth, life emerged rather early, approximately half billion years into its history or 4 billion years ago (Figure 12), although this number is quite uncertain. For about half of the history of life on Earth, only bacteria and archaea existed. Both groups of organisms are also summarized as "prokaryotic", a term derived from ancient Greek and meaning "before nut (or nucleus)" referring to the compartment containing the DNA that is found eukaryotes. Bacteria and archaea were and still are relatively simple, in that they consisted of single cells and do not contain specialized compartments like a nucleus or organelles. As the oldest groups of organisms, these prokaryotes evolved all kinds of metabolic capabilities that they used to harness energy from their environment as we will discuss in more detail in Chapter 6. Oxygenic photosynthesis stands out among these different energyharvesting mechanisms, as the prokaryotes earning their living with this particular mode of energy conversion found a way to split off electrons from water and liberate molecular oxygen (O2) as a byproduct. Over millions of years, the metabolic activities of tiny bacteria led to the accumulation of O2 in the atmosphere, starting a revolution in atmospheric chemistry: The Great Oxidation Event that began approximately 2.4 billion years ago and changed the appearance of our planet (Figure 12). This can be illustrated in two ways. First, O2 in the atmosphere reacted to yield the ozone (O3) shield in the upper atmosphere that protects the Earth’s surface from dangerous incoming ultraviolet (UV) light from the Sun. Thus, life outside the ocean would have been probably impossible without an ozone layer. Second, and arguably more fundamental. The rise of O2 paved the way for the evolution of a metabolism that 13 consumes O2 (aerobic metabolism), which is highly rewarding from a bioenergetic point of view. It is linked to the emergence of eukaryotic life forms that appeared around 2 billion years ago (the term eukaryote is again derived from ancient Greek and means "good nut" to highlight the feature of eukaryotes possessing a nucleus). Importantly also, the ability to breathe with O2 is linked to the emergence of complex life. Complex multicellular eukaryotes, such as plants and animals, evolved only much later around 0.5 billion (or 500 million) years ago, whereas the first modern humans appeared about 230,000 years ago. The complexity we see today would have been impossible without the O2 that we and other animals use and that reacts through biochemical processes with food to provide the energy we depend on. Figure 12. History of Earth with major biological innovations. While Bacteria and Archaea refer back to the origin of cellular life, eukarya evolved about 2 billion years ago (BYA). The oxygen concentration started accumulating after the great oxidation event after oxygenic photosynthesis that releases molecular oxygen from water has evolved in ancestors of extant Cyanobacteria. 14 Three domains of Life The evolutionary history of life on Earth (Figure 12) can be contextualized by coming back to the three domains of life that are currently the accepted: Bacteria, Archaea, and Eukarya. As previously stated, Eukarya are relatively young in comparison to the two prokaryotic groups, Bacteria and Archaea. The last universal common ancestor (LUCA) is a theoretical concept in evolutionary biology based on the biochemical similarities among all existing organisms. It thus refers to a common origin of cellular life from which it has diversified. It is sometimes depicted as a hypothetical single-celled organism. However, we do not know this, and it may be misleading to think of a singular organism as a physical entity that once existed. An alternative perspective is that LUCA may have been 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 and the ability to transcribe genomic information, DNA, into RNA and to translate RNA into proteins, as we will discuss in Chapter 4. These features, or basic biological macromolecules, which formed from simpler building blocks in the most distant past, are still present today, universally, in all living organisms, regardless of whether they are bacteria or archaea - or even a plant, or a human being. The central functions common to all cells can be seen as a kind of molecular fossil, a testimony of a shared evolutionary history of all life forms and of the relatedness of all living beings. A prominent characteristic of the two older prokaryotic groups, Bacteria and Archaea, is their remarkable chemical diversity in terms of energy metabolism (Figure 13): Although these little cells retain a relatively simple morphology and size, which is optimally suited for interaction with their environment, these microorganisms have evolved the ability to utilize a wide range of substrates or fuels. Figure 13. Unity and diversity of life. While bacteria and archaea feature an They can feed on a vast array extraordinary biochemical diversity in terms of energy metabolism, some eukarya evolved high morphological diversity as multicellular organisms, although in terms of different electron donors, of numbers many protists, unicellular eukaryotes exist as well. and as a result can form many different products, making them highly versatile in terms of their energy metabolism when considering all of them together. On the other hand, the Eukarya domain features a higher level of cellular complexity than prokaryotes (Box 2), being highly compartmentalized and requiring more regulatory checkpoints for basic tasks and for reproduction compared to prokaryotes. Eukaryotes have also evolved ways for tight cooperation and differentiation into 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 at the organismic level, which is evident in the wide range of forms and associated functions of eukaryotic organisms. 15 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 14), scientists such as Antonie van Leeuwenhoek and Robert Hooke began to discover tiny life forms, which today are the subject of the field of microbiology. Later on, 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 micro-organisms. For a long time, prokaryotes were considered as a single group of organisms. However, the advent of 14. First microscopes that revealed molecular methods enabled researchers to compare Figure microscopic life. Microscope developed by Antonie van Leeuwenhoek (1632-1722) (left), actual genetic sequences and the detailed structure microscope built by Hooke (middle). Color of cell components, revealing that prokaryotes in fact plate containing the eleven figures accompanying Robert Koch’s and Cohn’s papers from are composed of two distinct groups or domains of 1876. Original volume provided courtesy of the life: Bacteria and Archaea. Their separation goes far Farlow Botanical Library, Harvard University. back to the origin of cellular life. Box 2. Cells: the central units of biology (1-5 µm) (10-100 µm) Figure 15. Comparison of typical prokaryotic and eukaryotic cells. (taken from https://sciencenotes.org/prokaryotic-vseukaryotic-similarities-and-differences/) Cells form the central unit of life and are capable of independent reproduction. Prokaryotic cells typically measure about 1 to 2 µm in size and are thus much smaller than eukaryotic cells (Figure 15) that typically have a diameter of about 10 to 100 µm. Eukaryotic cells possess a nucleus and various other compartments, such as mitochondria, which we will examine later. For now, we will focus on simpler prokaryotic cells that started evolutionary history and dominate in terms of numbers on Earth, as discussed above (Figure 3). The common 16 perception of a prokaryotic cell as a membraneous bubble enclosing a reaction space with DNA and some proteins floating in a watery cytoplasm is misleading. The interior of a cell is quite densely packed (Figure 16), with biological macromolecules competing for space in the crowded molecular environment they form, much like a real estate market. Figure 16. Composition of a prokaryotic cell. (Phillips, Physical Biology of the Cell, 2nd e, Garland Sci , Fig. 2.4) The DNA content of the best-studied bacterium Escherichia coli amounts to 4.6 million base pairs. On average, a gene is about 1000 base pairs long, resulting in around 4,300 proteincoding genes in the model bacterium. These genes are expressed in varying amounts at different times, contributing to the highly dynamic nature of the proteome, which is the sum of all proteins at a given time. This dynamic nature is the result of the cell constantly adapting to internal signals and environmental cues. In fact, under well-defined laboratory conditions, only about 500 genes are required for minimal cell functions. While DNA only accounts for 3% of a bacterial cell’s dry weight, its more than three million protein molecules make for more than half of the cell dry weight (55%), after all the water is removed (Figure 17). Of these proteins, about two-thirds are located in the cytoplasm, inside the cell, with the remaining one-third embedded in the membrane. All RNA molecules together sum up to approximately 20% of the cell's dry weight, with the large majority being part of ribosomes, the complex super-molecular structures that translate mRNA into amino acid chains as we will discuss in Chapter 4. The cell membrane of E. coli is composed of more than 20 million phospholipid molecules, accounting for approximately 9% of the cell's dry weight. All these types of macromolecules have special Figure 17. Major cell components (model bacterium: properties that make them suitable for their Escherichia coli). Composition of an E. coli cell. Each function as catalysts to accelerate reactions, as polygon area represents the relative fraction of the corresponding constituent in the cell dry mass (Voroni structural components, and to separate outside diagram). (Milo & Phillips, Cell Biology by the from inside, which we will discuss in Chapter 3. Numbers, Taylors and Francis) 17 "Trees of Life" The impulse to classify living beings is perhaps as old as mankind itself. In 1735, Carl Linnaeus developed the first classification system based on morphological similarities between organisms. In his catalogue of living beings, 'Systema naturae', Linnaeus grouped the 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 observed in an evolutionary context. Consistent with the prevailing beliefs of his time, Linnaeus regarded living beings as "fixed" and unchanging. This view began to crumble as a result of several observations. Georges Cuvier was one of the first scientists to systematically study fossils, biological material that turned into stones, and is considered one of the founders of the science of paleontology. He had 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 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 laws and processes that shape the 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 the subject to constant change. Figure 18. Charles Darwin and Alfred R Wallace. This revolutionary thought was brought forward independently by Alfred Wallace and Charles Darwin (Figure 18), even if later on Darwin’s book On the Origin of Species by Means of Natural Selection (published in 1859) became much more widely known than Wallace’s paper. In his famous book, 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 conclusions about the past (Figure 19). As individuals compete for limited resources in the socalled "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 18 passed on to future generations than those of less environmentally adapted individuals, 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. Darwin, of course, had no understanding of how the evolutionary principles acted at the molecular level, since DNA was only discovered much later to encode the hereditary information. From today’s perspective, however, the Darwinian modifications in heritable traits correspond to random mutations in genetic sequences. These mutations occur by chance and are the source of genetic Figure 19. Inferring evolution using “top down” and “bottom up” approaches. variation, on which natural selection acts upon. Mutations are neither inherently good or bad, but depending on the environment, they may confer an advantage or disadvantage to their carrier or are simply neutral. In the process of natural selection, mutations might provide organisms a selective advantage allowing them to occupy a new niche or outcompete others, thus benefitting from the innovation step we discussed above (Figure 7). As a result, these advantages are preserved in the genome and passed on through generations, and additional ones can occur, leading eventually to entirely new lineages of organisms. Nowadays evolution can be observed in laboratory experiments in real time under our eyes, for example by following the gradual appearance of resistance towards an antibiotic by the bacterium Escherichia coli (Figure 19). This way of observing the evolutionary dynamics in live action allows scientists to trace the sequence of events that lead to an observed outcome, a phenotype, by tracing changes in the genome, the genotype. However, we cannot go back in time. If we want to infer the evolutionary history and the relationships between living species, a reverse approach is needed by which we look backwards into the past. That is the scope and aim of phylogenetic classification systems. Over the last 100 years, a large variety of different systems – or "Trees of Life" – have been proposed. This shows that not only do living organisms evolve over time, but so do the ideas about the relationships between them. Ernst Haeckel proposed the concept of a monophyletic tree of life, in which all life forms share a common ancestor, and their evolutionary relationships are represented as branches, as he famously illustrated in his "oak tree of life" from 1866 (Figure 20). His tree of life and others that followed had limitations, as they were based on a limited number of observable characteristics, mostly morphological. 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). An 19 example of this is the ability to fly. In contrast to earlier beliefs, we now know that birds, bats, and insects independently evolved the ability to fly. Figure 20. Oak tree of life developed and drawn by Ernst Haeckel (18341919). The tree illustrates the monophyletic origin and represents the first phylogentic tree of life, published in 1866. Source: Ernst Haeckel, Allgemeine Entwicklungsgeschichte der Organismen, Berlin 1866; Library University of Darmstadt, Germany. But what kind of evidence can shed light on the largely unknown evolutionary paths from the past? The answer is molecular data. Genetic information has been passed on from generation to generation and mutations occur, some of which become fixed in populations over time. We can therefore conclude that organisms are more distantly related the more different their genetic sequences are. Conversely, two organisms sharing similar genetic material are more closely related, with some caveats that will be discussed below. Starting in the late 1970s, Carl Woese and his colleagues applied this logic and concentrated on the sequences of ribosomal RNA (rRNA) 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 can only occur slowly over time as most changes are penalized, resulting in non-viable offspring. 20 However, despite the slow rate of change of the molecules that make up the ribosome as a whole, their genetic sequences contain enough variable parts to provide deeper insights into evolutionary relationships. In 1990, Woese and his colleagues published a paper about the "natural systems of organisms", in which they proposed to split the prokaryotes into two different domains, Archaea and Bacteria, resulting in three domains tree of life (Figure 21), now also known as the Woese tree. "Molecular comparisons show that life on this planet divides into three primary groupings", they write and conclude 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 rRNA phylogenetic tree, they are just a part of the overall diversity of living organisms and in contemporary representations, they form little sub-branches at the edge of the domain Eukarya (Figure 22). Figure 21. The phylogenetic tree of life based on ribosomal information as a molecular marker. Life on this planet can be divided into three primary groupings or domains, the Bacteria, the Archaea and the Eucarya (modified from Stetter 1996, modified from Woese et al. 1990) The length of the branches represents the phylogenetic distance of the groups. Figure 22. Updated «tree of life» . Red dots indicate microbial groups for which no representative has been cultured yet. Discover Magazine, Redrawn after Hug et al. 2016 Nature Microbiology 1, 16048. This three-domain view is still accepted today. However, when other scientists started to look at the sequences of other evolutionarily conserved genes, they realized that the genome is structured like a mosaic. It means that each gene, which encode each a protein with a certain function, has its own history. When looking at all genes encoded in the genomes of presentday Eukarya, scientists noted that they contain genes that are more closely related to sequences from extant Archaea and other genes that are clearly related to sequences found in extant Bacteria. The explanation for these findings is that Eukarya are chimeric, i.e., derived from different organisms. Several researchers, including Konstantin Mereschkowski, had laid the foundation to conceptualize the origin of the chimeric structure of Eukaryotes in 1910 by postulating symbiogenesis, that eukaryotic cells are the result of symbiotic relationships. Based on this 21 work and on later discoveries showing that chloroplasts and mitochondria both contain their own DNA, 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 aerobic respiring bacteria, which were stably incorporated into an Archaeon and that (2) chloroplasts are the result of the incorporation of a cyanobacterium into a eukaryotic cell. Latest sequencing data suggest a bacterium most closely related to extant alphaproteobacteria as the origin of mitochondria and so-called Asgard archaea as the closest relative of the original host. In consequence, considering the ribosomal tree, Eukaryotes emerged from within the domain or Archaea. These endosymbiotic events require that the tree of life can be drawn in a different way (Figure 23). 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 living beings on Earth for more than a billion years, as we 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 FECA, the first eukaryotic common ancestor, that evolved further giving rise to LECA, the last eukaryotic common ancestor and the beginning of the domain Eukarya. The latter from there on existed and still do live in parallel to bacteria and archaea, and often in symbiotic relationships. Figure 23. A modern view of the "Tree of Life": Two different prokaryotic groups evolved from LUCA, the last universal common ancestor. A billion years later, when a bacterial cell entered an archaeal cell, LECA, the last eukaryotic common ancestor, was born. This marks the beginning of the domain Eukarya. (Modified from Martin et al. (2017) MMBR 81:e00008-17) Endosymbiosis explains how eukaryotes are of chimeric nature with different genes most closely related to their counterparts in either bacteria or archaea, marking a monophyletic origin of Eukarya (Figure 23). Chimerism, as it occurs through endosymbiosis, can be considered a tremendous "speed booster" in the evolution of life that was accompanied by the emergence of sexual reproduction in eukaryotes, meiosis, as a built-in program to merge two cells with their entire genomes. As we said above, after the emergence of the mitochondria 22 another endosymbiotic event occurred, resulting in the formation of chloroplasts in a branch of Eukarya, leading to the emergence of algae and plants. Specifically, it is the plants in terrestrial systems that constitute most of the biomass on Earth today (Figure 3) and bacteria that continued to dominate the living world in terms of number of organisms (Figure 4). Let’s now come back to chimerism. It occurred at the origin of eukaryotes and it is also manifested as a result of sexual reproduction, when the genomes of two organisms are mixed in one cell to give rise to offspring. However, chimerism is also central to prokaryotes, and likely from the beginning. The genomes of extant organisms attest chimerism as the consequence of incorporation of foreign DNA pieces into genomes - in other words, as a consequence of horizontal gene transfer (HGT). Instead of mixing and matching entire genomes, pieces of DNA can be transferred through various mechanisms including viruses or 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. Key points: ▪ ▪ ▪ ▪ Earth provided the conditions and elements for life to emerge, and life in turn had profound effects on Earth Bacteria and archaea were the only type of organisms that existed until halfway through Earth’s history before eukaryotes emerged Sequence information for ribosomes is commonly used to infer relatedness of all organisms to each other Beyond mutations that cause changes in genomes, bacteria and archaea exchange genetic material independently from reproduction, while in eukaryotes reproduction involves the combining entire genomes from two different 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) Why do some scientists say that the ribosomal tree is the "tree of 1%"? 23 Origin of Life Now that we have looked at the composition of biomass on Earth today (Figure 3), the history of life on our planet, the planetary conditions and seen that species went also extinct, we would like to go back to the beginnings of life on Earth and discuss some of the ideas that have been put forward. Inherently, the topic is difficult from a scientific perspective due to the lack of reproducibility. We begin with a historical consideration of how the problem has been approached. From the time of Aristotle and the ancient Greeks until the 17th century, life was generally believed to have arisen from nonliving matter, such as rats and snakes having formed from the mud of the Nile. But in 1668, Francesco Redi put the idea of spontaneous generation to the test by placing pieces of meat in different open or sealed containers. Because of the inclusion of a control condition in this simple setup, Redi is considered as one of the founders of experimental biology. 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’s conclusion was that maggots could not spontaneously emerge out of the rotting meat. He summarized his findings as the famous phrase omne vivum ex vivo, which in Latin means "all life [is] from life." Almost 200 years later, Louis Pasteur, after whom ‘pasteurization’ is named for sterilizing material, conducted what is known as the swan-neck bottle experiment to test that smaller life forms, microorganisms, arise from others and not spontaneously. His experimental set up involved long curved tubes through which air could enter, but where dust and air-borne microbes would remain trapped in the bend. In fact, after filling the bottles with meat broth and sterilizing the liquid by heating, Pasteur saw that the liquid remained sterile – and only started to be populated by microorganisms when the bottles were tipped and the liquid entered into contact with the trapped microbes in the bend. From his experiment Pasteur concluded that there were no living beings coming into the world without parents similar to themselves. Although this rule is still pertinent today, it leaves one big question unanswered: How did life start in the first place? How can inanimate matter evolve into a complex system that uses energy from its environment to grow and replicate itself? Essential conditions For the origin of life quite a number of hypotheses have been put forward and are heavily debated. There is currently no consensus. There is also no consensus whether the emergence of cellular life is deterministic or predictable, in principle - or rather the result of chance as the outcome of many random events. However, there is more agreement on conditions that must be met or were central for the transition from geochemistry to the first cellular life as we saw above (Figure 9), the transition from non-living to living entities: 24 - - - Some sort of catalysts must have promoted the abiotic synthesis of small organic molecules. This opens up the possibility of self-organizing networks of chemical reactions that operate as autocatalytic cycles. There must have been a stable source of energy that enabled the evolving pre-biotic systems to polymerize small molecular units and to maintain a state far from thermodynamic equilibrium. There also must have been some sort of barriers that permitted physical compartmentalization, a separation of an inside from an outside. 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, waste by-products of these networks must have been continuously removed to the outside. This barrier should not be sealing the interior completely off its environment, as matter need to enter from outside also. Among scientists, there is considerable debate about the sequence of events that led to the formation of protocells, and not only how it occurred but also where. Some argue that metabolism came first with catalysts playing a key role in establishing autocatalytic metabolic networks in the beginning, while others place greater emphasis on membranes or hereditary molecules such as RNA. The problem is that we do not know of any intermediate steps and we cannot study any protocells or primitive cells, because even the simplest unicellular organisms known today are complex systems. They already present all the various features (metabolism, membranes, hereditary molecules) that are characteristic of every living being on Earth. So far, life has also not been formed "de novo" under laboratory conditions and even if it would be one day, we do not know whether this is how life emerged on Earth as the start of life on Earth is lost in history. There are two different and complementary ways to approach the origin of life (Figure 24). The first – or bottom-up – approach consists in studying geochemical processes and deriving scenarios that could have contributed to the formation of more and more complex systems. The second – or top-down – approach consists of comparing today’s diversity of organisms and trying to infer their common roots. This information can then be used to make educated guesses 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. Figure 24. Two fundamental different views to approach the origins of life. 25 Scenarios for the transition from geochemistry to biochemistry and the origin of cellular life Although Darwin did not write about the beginning of life in his On the Origin of Species, he later speculated in a letter to his closest friend that life may have started in a "warm little pond." Similarly, Alexander Oparin and John Haldane proposed in the 1920s that inorganic compounds (such as carbon dioxide, ammonia, and water vapor) contained in the anoxic atmosphere of the early Earth reacted with each other and, by a slow process of molecular evolution, gave rise to more and more complex organic molecules that over time accumulated in the primitive oceans. This "primordial soup" theory gained considerable traction in 1953 when Stanley Miller and Harold Urey demonstrated with a now-famous experiment that inorganic precursors could indeed be transformed into organic molecules such as amino acids and other basic building blocks of major biochemical polymers. Miller and Urey used electric discharges that simulated natural lightning as the source of energy to drive the reactions. Thus, by providing evidence that important steps in the prebiotic chemical evolution could take place under natural conditions, the Miller-Urey experiment turned the "primordial soup" hypothesis into an explanation for the possible origin of life that