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Discuss the diversity of prokaryotic cells
In the recent past, scientists grouped living things into five kingdoms—animals, plants, fungi, protists, and prokaryotes—based on several criteria, such as the absence or presence of a nucleus and other membrane-bound organelles, the absence or presence of cell walls, multicellularity, and so on. The domain Bacteria comprises all organisms in the kingdom Bacteria, the domain Archaea comprises the rest of the prokaryotes, and the domain Eukarya comprises all eukaryotes—including organisms in the kingdoms Animalia, Plantae, Fungi, and Protista.
Two of the three domains—Bacteria and Archaea—are prokaryotic. Prokaryotes were the first inhabitants on Earth, appearing 3.5 to 3.8 billion years ago. These organisms are abundant and ubiquitous; that is, they are present everywhere. In addition to inhabiting moderate environments, they are found in extreme conditions: from boiling springs to permanently frozen environments in Antarctica; from salty environments like the Dead Sea to environments under tremendous pressure, such as the depths of the ocean; and from areas without oxygen, such as a waste management plant, to radioactively contaminated regions, such as Chernobyl. Prokaryotes reside in the human digestive system and on the skin, are responsible for certain illnesses, and serve an important role in the preparation of many foods.
What You’ll Learn to Do
- Describe the evolutionary history of prokaryotes
- Discuss the distinguishing features of extremophiles
- Understand why it is difficult to culture prokaryotes
- Discuss why prokaryotes often form biofilms
The learning activities for this section include the following:
- Evolutionary History of Prokaryotes
- Life in Moderate and Extreme Environments
- Culturing Prokaryotes
- Prokaryotic Biofilms
- Self Check: Prokaryotic Diversity
Exploring prokaryotic diversity in the genomic era
Our understanding of prokaryote biology from study of pure cultures and genome sequencing has been limited by a pronounced sampling bias towards four bacterial phyla - Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes - out of 35 bacterial and 18 archaeal phylum-level lineages. This bias is beginning to be rectified by the use of phylogenetically directed isolation strategies and by directly accessing microbial genomes from environmental samples.
It is a common misconception that microorganisms isolated in pure culture from an environment represent the numerically dominant and/or functionally significant species in that environment. In fact, microorganisms isolated using standard cultivation methods are rarely numerically dominant in the communities from which they were obtained: instead, they are isolated by virtue of their ability to grow rapidly into colonies on high-nutrient artificial growth media, typically under aerobic conditions, at moderate temperatures. Easily isolated organisms are the 'weeds' of the microbial world and are estimated to constitute less than 1% of all microbial species (this figure was estimated by comparing plate counts with direct microscopic counts of microorganisms in environmental samples it has been called the "great plate-count anomaly" ).
Given that the study of a microorganism is simpler if you have it in pure culture on an agar plate, it is not surprising that most of what we know about microbiology comes from the study of microbial weeds. For example, approximately 65% of published microbiological research from 1991 to 1997 was dedicated to only eight bacterial genera, Escherichia (18%), Helicobacter (8%), Pseudomonas (7%), Bacillus (7%), Streptococcus (6%), Mycobacterium (6%), Staphylococcus (6%) and Salmonella (5%) , all of which are relatively simple to grow on agar plates. Intuitively, it seems unlikely that this handful of organisms can be representative of the approximately 5,000 validly described prokaryotic species , but exactly how unrepresentative are they? And if more than 99% of microorganisms in the environment are unculturable using standard techniques, how representative are cultivated microorganisms of prokaryotic diversity as a whole? To answer these questions, we need a framework for placing prokaryotic species and genera in a broader evolutionary context.
13.1 Prokaryotic Diversity
Prokaryotes are present everywhere. They cover every imaginable surface where there is sufficient moisture, and they live on and inside of other living things. There are more prokaryotes inside and on the exterior of the human body than there are human cells in the body. Some prokaryotes thrive in environments that are inhospitable for most other living things. Prokaryotes recycle nutrients—essential substances (such as carbon and nitrogen)—and they drive the evolution of new ecosystems, some of which are natural while others are man-made. Prokaryotes have been on Earth since long before multicellular life appeared.
The advent of DNA sequencing provided immense insight into the relationships and origins of prokaryotes that were not possible using traditional methods of classification. A major insight identified two groups of prokaryotes that were found to be as different from each other as they were from eukaryotes. This recognition of prokaryotic diversity forced a new understanding of the classification of all life and brought us closer to understanding the fundamental relationships of all living things, including ourselves.
Early Life on Earth
When and where did life begin? What were the conditions on Earth when life began? Prokaryotes were the first forms of life on Earth, and they existed for billions of years before plants and animals appeared. Earth is about 4.54 billion years old. This estimate is based on evidence from the dating of meteorite material, since surface rocks on Earth are not as old as Earth itself. Most rocks available on Earth have undergone geological changes that make them younger than Earth itself. Some meteorites are made of the original material in the solar disk that formed the objects of the solar system, and they have not been altered by the processes that altered rocks on Earth. Thus, the age of meteorites is a good indicator of the age of the formation of Earth. The original estimate of 4.54 billion years was obtained by Clare Patterson in 1956. His meticulous work has since been corroborated by ages determined from other sources, all of which point to an Earth age of about 4.54 billion years.
Early Earth had a very different atmosphere than it does today. Evidence indicates that during the first 2 billion years of Earth’s existence, the atmosphere was anoxic , meaning that there was no oxygen. Therefore, only those organisms that can grow without oxygen— anaerobic organisms—were able to live. Organisms that convert solar energy into chemical energy are called phototrophs . Phototrophic organisms that required an organic source of carbon appeared within one billion years of the formation of Earth. Then, cyanobacteria , also known as blue-green algae, evolved from these simple phototrophs one billion years later. Cyanobacteria are able to use carbon dioxide as a source of carbon. Cyanobacteria (Figure 13.2) began the oxygenation of the atmosphere. The increase in oxygen concentration allowed the evolution of other life forms.
Before the atmosphere became oxygenated, the planet was subjected to strong radiation thus, the first organisms would have flourished where they were more protected, such as in ocean depths or beneath the surface of Earth. At this time, too, strong volcanic activity was common on Earth, so it is likely that these first organisms—the first prokaryotes—were adapted to very high temperatures. These are not the typical temperate environments in which most life flourishes today thus, we can conclude that the first organisms that appeared on Earth likely were able to withstand harsh conditions.
Microbial mats may represent the earliest forms of life on Earth, and there is fossil evidence of their presence, starting about 3.5 billion years ago. A microbial mat is a large biofilm, a multi-layered sheet of prokaryotes (Figure 13.3a), including mostly bacteria, but also archaea. Microbial mats are a few centimeters thick, and they typically grow on moist surfaces. Their various types of prokaryotes carry out different metabolic pathways, and for this reason, they reflect various colors. Prokaryotes in a microbial mat are held together by a gummy-like substance that they secrete.
The first microbial mats likely obtained their energy from hydrothermal vents. A hydrothermal vent is a fissure in Earth’s surface that releases geothermally heated water. With the evolution of photosynthesis about 3 billion years ago, some prokaryotes in microbial mats came to use a more widely available energy source—sunlight—whereas others were still dependent on chemicals from hydrothermal vents for food.
Fossilized microbial mats represent the earliest record of life on Earth. A stromatolite is a sedimentary structure formed when minerals are precipitated from water by prokaryotes in a microbial mat (Figure 13.3b). Stromatolites form layered rocks made of carbonate or silicate. Although most stromatolites are artifacts from the past, there are places on Earth where stromatolites are still forming. For example, living stromatolites have been found in the Anza-Borrego Desert State Park in San Diego County, California.
Some prokaryotes are able to thrive and grow under conditions that would kill a plant or animal. Bacteria and archaea that grow under extreme conditions are called extremophiles , meaning “lovers of extremes.” Extremophiles have been found in extreme environments of all kinds, including the depths of the oceans, hot springs, the Arctic and the Antarctic, very dry places, deep inside Earth, harsh chemical environments, and high radiation environments. Extremophiles give us a better understanding of prokaryotic diversity and open up the possibility of the discovery of new therapeutic drugs or industrial applications. They have also opened up the possibility of finding life in other places in the solar system, which have harsher environments than those typically found on Earth. Many of these extremophiles cannot survive in moderate environments.
Concepts in Action
Watch a video showing the Director of the Planetary Science Division of NASA discussing the implications that the existence extremophiles on Earth have on the possibility of finding life on other planets in our solar system, such as Mars.
Until a couple of decades ago, microbiologists thought of prokaryotes as isolated entities living apart. This model, however, does not reflect the true ecology of prokaryotes, most of which prefer to live in communities where they can interact. A biofilm is a microbial community held together in a gummy-textured matrix, consisting primarily of polysaccharides secreted by the organisms, together with some proteins and nucleic acids. Biofilms grow attached to surfaces. Some of the best-studied biofilms are composed of prokaryotes, although fungal biofilms have also been described.
Biofilms are present almost everywhere. They cause the clogging of pipes and readily colonize surfaces in industrial settings. They have played roles in recent, large-scale outbreaks of bacterial contamination of food. Biofilms also colonize household surfaces, such as kitchen counters, cutting boards, sinks, and toilets.
Interactions among the organisms that populate a biofilm, together with their protective environment, make these communities more robust than are free-living, or planktonic, prokaryotes. Overall, biofilms are very difficult to destroy, because they are resistant to many of the common forms of sterilization.
Characteristics of Prokaryotes
There are many differences between prokaryotic and eukaryotic cells. However, all cells have four common structures: a plasma membrane that functions as a barrier for the cell and separates the cell from its environment cytoplasm, a jelly-like substance inside the cell genetic material (DNA and RNA) and ribosomes, where protein synthesis takes place. Prokaryotes come in various shapes, but many fall into three categories: cocci (spherical), bacilli (rod-shaped), and spirilla (spiral-shaped) (Figure 13.4).
The Prokaryotic Cell
Recall that prokaryotes (Figure 13.5) are unicellular organisms that lack organelles surrounded by membranes. Therefore, they do not have a nucleus but instead have a single chromosome—a piece of circular DNA located in an area of the cell called the nucleoid. Most prokaryotes have a cell wall lying outside the plasma membrane. The composition of the cell wall differs significantly between the domains Bacteria and Archaea (and their cell walls also differ from the eukaryotic cell walls found in plants and fungi.) The cell wall functions as a protective layer and is responsible for the organism’s shape. Some other structures are present in some prokaryotic species, but not in others. For example, the capsule found in some species enables the organism to attach to surfaces and protects it from dehydration. Some species may also have flagella (singular, flagellum) used for locomotion, and pili (singular, pilus) used for attachment to surfaces and to other bacteria for conjugation. Plasmids, which consist of small, circular pieces of DNA outside of the main chromosome, are also present in many species of bacteria.
Both Bacteria and Archaea are types of prokaryotic cells. They differ in the lipid composition of their cell membranes and in the characteristics of their cell walls. Both types of prokaryotes have the same basic structures, but these are built from different chemical components that are evidence of an ancient separation of their lineages. The archaeal plasma membrane is chemically different from the bacterial membrane some archaeal membranes are lipid monolayers instead of phosopholipid bilayers.
The Cell Wall
The cell wall is a protective layer that surrounds some prokaryotic cells and gives them shape and rigidity. It is located outside the cell membrane and prevents osmotic lysis (bursting caused by increasing volume). The chemical compositions of the cell walls vary between Archaea and Bacteria, as well as between bacterial species. Bacterial cell walls contain peptidoglycan , composed of polysaccharide chains cross-linked to peptides. Bacteria are divided into two major groups: Gram-positive and Gram-negative , based on their reaction to a procedure called Gram staining. The different bacterial responses to the staining procedure are caused by cell wall structure. Gram-positive organisms have a thick wall consisting of many layers of peptidoglycan. Gram-negative bacteria have a thinner cell wall composed of a few layers of peptidoglycan and additional structures, surrounded by an outer membrane (Figure 13.6).
Which of the following statements is true?
- Gram-positive bacteria have a single cell wall formed from peptidoglycan.
- Gram-positive bacteria have an outer membrane.
- The cell wall of Gram-negative bacteria is thick, and the cell wall of Gram-positive bacteria is thin.
- Gram-negative bacteria have a cell wall made of peptidoglycan, while Gram-positive bacteria have a cell wall made of phospholipids.
Archaeal cell walls do not contain peptidoglycan. There are four different types of archaeal cell walls. One type is composed of pseudopeptidoglycan . The other three types of cell walls contain polysaccharides, glycoproteins, and surface-layer proteins known as S-layers.
Reproduction in prokaryotes is primarily asexual and takes place by binary fission. Recall that the DNA of a prokaryote exists usually as a single, circular chromosome. Prokaryotes do not undergo mitosis. Rather, the chromosome loop is replicated, and the two resulting copies attached to the plasma membrane move apart as the cell grows in a process called binary fission. The prokaryote, now enlarged, is pinched inward at its equator, and the two resulting cells, which are clones, separate. Binary fission does not provide an opportunity for genetic recombination, but prokaryotes can alter their genetic makeup in three ways.
Binary fission as a way of reproduction does not provide an opportunity for genetic recombination and increased genetic variability. However, prokaryotes can alter their genetic makeup by three mechanisms of obtaining exogenous DNA. In a process called transformation , the cell takes in DNA found in its environment that is shed by other prokaryotes, alive or dead. A pathogen is an organism that causes a disease. If a nonpathogenic bacterium takes up DNA from a pathogen and incorporates the new DNA in its own chromosome, it too may become pathogenic. In transduction , bacteriophages, the viruses that infect bacteria, move DNA from one bacterium to another. Archaea have a different set of viruses that infect them and translocate genetic material from one individual to another. During conjugation , DNA is transferred from one prokaryote to another by means of a pilus that brings the organisms into contact with one another. The DNA transferred is usually a plasmid, but parts of the chromosome can also be moved.
Cycles of binary fission can be very rapid, on the order of minutes for some species. This short generation time coupled with mechanisms of genetic recombination result in the rapid evolution of prokaryotes, allowing them to respond to environmental changes (such as the introduction of an antibiotic) very quickly.
How Prokaryotes Obtain Energy and Carbon
Prokaryotes are metabolically diverse organisms. Prokaryotes fill many niches on Earth, including being involved in nutrient cycles such as the nitrogen and carbon cycles, decomposing dead organisms, and growing and multiplying inside living organisms, including humans. Different prokaryotes can use different sources of energy to assemble macromolecules from smaller molecules. Phototrophs obtain their energy from sunlight. Chemotrophs obtain their energy from chemical compounds.
Bacterial Diseases in Humans
Devastating pathogen-borne diseases and plagues, both viral and bacterial in nature, have affected and continue to affect humans. It is worth noting that all pathogenic prokaryotes are Bacteria there are no known pathogenic Archaea in humans or any other organism. Pathogenic organisms evolved alongside humans. In the past, the true cause of these diseases was not understood, and some cultures thought that diseases were a spiritual punishment or were mistaken about material causes. Over time, people came to realize that staying apart from afflicted persons, improving sanitation, and properly disposing of the corpses and personal belongings of victims of illness reduced their own chances of getting sick.
There are records of infectious diseases as far back as 3,000 B.C. A number of significant pandemics caused by Bacteria have been documented over several hundred years. Some of the largest pandemics led to the decline of cities and cultures. Many were zoonoses that appeared with the domestication of animals, as in the case of tuberculosis. A zoonosis is a disease that infects animals but can be transmitted from animals to humans.
Infectious diseases remain among the leading causes of death worldwide. Their impact is less significant in many developed countries, but they are important determiners of mortality in developing countries. The development of antibiotics did much to lessen the mortality rates from bacterial infections, but access to antibiotics is not universal, and the overuse of antibiotics has led to the development of resistant strains of bacteria. Public sanitation efforts that dispose of sewage and provide clean drinking water have done as much or more than medical advances to prevent deaths caused by bacterial infections.
In 430 B.C., the plague of Athens killed one-quarter of the Athenian troops that were fighting in the Great Peloponnesian War. The disease killed a quarter of the population of Athens in over 4 years and weakened Athens’ dominance and power. The source of the plague may have been identified recently when researchers from the University of Athens were able to analyze DNA from teeth recovered from a mass grave. The scientists identified nucleotide sequences from a pathogenic bacterium that causes typhoid fever. 1
From 541 to 750 A.D., an outbreak called the plague of Justinian (likely a bubonic plague) eliminated, by some estimates, one-quarter to one-half of the human population. The population in Europe declined by 50 percent during this outbreak. Bubonic plague would decimate Europe more than once.
One of the most devastating pandemics was the Black Death (1346 to 1361), which is believed to have been another outbreak of bubonic plague caused by the bacterium Yersinia pestis. This bacterium is carried by fleas living on black rats. The Black Death reduced the world’s population from an estimated 450 million to about 350 to 375 million. Bubonic plague struck London hard again in the mid-1600s. There are still approximately 1,000 to 3,000 cases of plague globally each year. Although contracting bubonic plague before antibiotics meant almost certain death, the bacterium responds to several types of modern antibiotics, and mortality rates from plague are now very low.
Concepts in Action
Watch a video on the modern understanding of the Black Death (bubonic plague) in Europe during the fourteenth century.
Over the centuries, Europeans developed resistance to many infectious diseases. However, European conquerors brought disease-causing bacteria and viruses with them when they reached the Western hemisphere, triggering epidemics that completely devastated populations of Native Americans (who had no natural resistance to many European diseases).
The Antibiotic Crisis
The word antibiotic comes from the Greek anti, meaning “against,” and bios, meaning “life.” An antibiotic is an organism-produced chemical that is hostile to the growth of other organisms. Today’s news and media often address concerns about an antibiotic crisis. Are antibiotics that were used to treat bacterial infections easily treatable in the past becoming obsolete? Are there new “superbugs”—bacteria that have evolved to become more resistant to our arsenal of antibiotics? Is this the beginning of the end of antibiotics? All of these questions challenge the healthcare community.
One of the main reasons for resistant bacteria is the overuse and incorrect use of antibiotics, such as not completing a full course of prescribed antibiotics. The incorrect use of an antibiotic results in the natural selection of resistant forms of bacteria. The antibiotic kills most of the infecting bacteria, and therefore only the resistant forms remain. These resistant forms reproduce, resulting in an increase in the proportion of resistant forms over non-resistant ones.
Another problem is the excessive use of antibiotics in livestock. The routine use of antibiotics in animal feed promotes bacterial resistance as well. In the United States, 70 percent of the antibiotics produced are fed to animals. The antibiotics are not used to prevent disease, but to enhance production of their products.
Concepts in Action
Watch an overview report on the problem of routine antibiotic administration to livestock and antibiotic-resistant bacteria.
Staphylococcus aureus, often called “staph,” is a common bacterium that can live in and on the human body, which usually is easily treatable with antibiotics. A very dangerous strain, however, has made the news over the past few years (Figure 13.7). This strain, methicillin-resistant Staphylococcus aureus (MRSA), is resistant to many commonly used antibiotics, including methicillin, amoxicillin, penicillin, and oxacillin. While MRSA infections have been common among people in healthcare facilities, it is appearing more commonly in healthy people who live or work in dense groups (like military personnel and prisoners). The Journal of the American Medical Association reported that, among MRSA-afflicted persons in healthcare facilities, the average age is 68 years, while people with “community-associated MRSA” (CA-MRSA) have an average age of 23 years. 2
In summary, society is facing an antibiotic crisis. Some scientists believe that after years of being protected from bacterial infections by antibiotics, we may be returning to a time in which a simple bacterial infection could again devastate the human population. Researchers are working on developing new antibiotics, but few are in the drug development pipeline, and it takes many years to generate an effective and approved drug.
Prokaryotes are everywhere: They readily colonize the surface of any type of material, and food is not an exception. Outbreaks of bacterial infection related to food consumption are common. A foodborne disease (colloquially called “food poisoning”) is an illness resulting from the consumption of food contaminated with pathogenic bacteria, viruses, or other parasites. Although the United States has one of the safest food supplies in the world, the Center for Disease Control and Prevention (CDC) has reported that “76 million people get sick, more than 300,000 are hospitalized, and 5,000 Americans die each year from foodborne illness.” 3
The characteristics of foodborne illnesses have changed over time. In the past, it was relatively common to hear about sporadic cases of botulism , the potentially fatal disease produced by a toxin from the anaerobic bacterium Clostridium botulinum. A can, jar, or package created a suitable anaerobic environment where Clostridium could grow. Proper sterilization and canning procedures have reduced the incidence of this disease.
Most cases of foodborne illnesses are now linked to produce contaminated by animal waste. For example, there have been serious, produce-related outbreaks associated with raw spinach in the United States and with vegetable sprouts in Germany (Figure 13.8). The raw spinach outbreak in 2006 was produced by the bacterium E. coli strain O157:H7. Most E. coli strains are not particularly dangerous to humans, (indeed, they live in our large intestine), but O157:H7 is potentially fatal.
All types of food can potentially be contaminated with harmful bacteria of different species. Recent outbreaks of Salmonella reported by the CDC occurred in foods as diverse as peanut butter, alfalfa sprouts, and eggs.
Epidemiology is the study of the occurrence, distribution, and determinants of health and disease in a population. It is, therefore, related to public health. An epidemiologist studies the frequency and distribution of diseases within human populations and environments.
Epidemiologists collect data about a particular disease and track its spread to identify the original mode of transmission. They sometimes work in close collaboration with historians to try to understand the way a disease evolved geographically and over time, tracking the natural history of pathogens. They gather information from clinical records, patient interviews, and any other available means. That information is used to develop strategies and design public health policies to reduce the incidence of a disease or to prevent its spread. Epidemiologists also conduct rapid investigations in case of an outbreak to recommend immediate measures to control it.
Epidemiologists typically have a graduate-level education. An epidemiologist often has a bachelor’s degree in some field and a master’s degree in public health (MPH). Many epidemiologists are also physicians (and have an MD) or they have a PhD in an associated field, such as biology or epidemiology.
Not all prokaryotes are pathogenic. On the contrary, pathogens represent only a very small percentage of the diversity of the microbial world. In fact, our life and all life on this planet would not be possible without prokaryotes.
Prokaryotes, and Food and Beverages
According to the United Nations Convention on Biological Diversity, biotechnology is “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.” 4 The concept of “specific use” involves some sort of commercial application. Genetic engineering, artificial selection, antibiotic production, and cell culture are current topics of study in biotechnology. However, humans have used prokaryotes to create products before the term biotechnology was even coined. And some of the goods and services are as simple as cheese, yogurt, sour cream, vinegar, cured sausage, sauerkraut, and fermented seafood that contains both bacteria and archaea (Figure 13.9).
Cheese production began around 4,000 years ago when humans started to breed animals and process their milk. Evidence suggests that cultured milk products, like yogurt, have existed for at least 4,000 years.
Using Prokaryotes to Clean up Our Planet: Bioremediation
Probably one of the most useful and interesting examples of the use of prokaryotes for bioremediation purposes is the cleanup of oil spills. The importance of prokaryotes to petroleum bioremediation has been demonstrated in several oil spills in recent years, such as the Exxon Valdez spill in Alaska (1989) (Figure 13.10), the Prestige oil spill in Spain (2002), the spill into the Mediterranean from a Lebanon power plant (2006,) and more recently, the BP oil spill in the Gulf of Mexico (2010). To clean up these spills, bioremediation is promoted by adding inorganic nutrients that help bacteria already present in the environment to grow. Hydrocarbon-degrading bacteria feed on the hydrocarbons in the oil droplet, breaking them into inorganic compounds. Some species, such as Alcanivorax borkumensis, produce surfactants that solubilize the oil, while other bacteria degrade the oil into carbon dioxide. In the case of oil spills in the ocean, ongoing, natural bioremediation tends to occur, inasmuch as there are oil-consuming bacteria in the ocean prior to the spill. Under ideal conditions, it has been reported that up to 80 percent of the nonvolatile components in oil can be degraded within 1 year of the spill. Other oil fractions containing aromatic and highly branched hydrocarbon chains are more difficult to remove and remain in the environment for longer periods of time. Researchers have genetically engineered other bacteria to consume petroleum products indeed, the first patent application for a bioremediation application in the U.S. was for a genetically modified oil-eating bacterium.
Prokaryotes in and on the Body
Humans are no exception when it comes to forming symbiotic relationships with prokaryotes. We are accustomed to thinking of ourselves as single organisms, but in reality, we are walking ecosystems. There are 10 to 100 times as many bacterial and archaeal cells inhabiting our bodies as we have cells in our bodies. Some of these are in mutually beneficial relationships with us, in which both the human host and the bacterium benefit, while some of the relationships are classified as commensalism , a type of relationship in which the bacterium benefits and the human host is neither benefited nor harmed.
Human gut flora lives in the large intestine and consists of hundreds of species of bacteria and archaea, with different individuals containing different species mixes. The term “flora,” which is usually associated with plants, is traditionally used in this context because bacteria were once classified as plants. The primary functions of these prokaryotes for humans appear to be metabolism of food molecules that we cannot break down, assistance with the absorption of ions by the colon, synthesis of vitamin K, training of the infant immune system, maintenance of the adult immune system, maintenance of the epithelium of the large intestine, and formation of a protective barrier against pathogens.
The surface of the skin is also coated with prokaryotes. The different surfaces of the skin, such as the underarms, the head, and the hands, provide different habitats for different communities of prokaryotes. Unlike with gut flora, the possible beneficial roles of skin flora have not been well studied. However, the few studies conducted so far have identified bacteria that produce antimicrobial compounds as probably responsible for preventing infections by pathogenic bacteria.
Researchers are actively studying the relationships between various diseases and alterations to the composition of human microbial flora. Some of this work is being carried out by the Human Microbiome Project, funded in the United States by the National Institutes of Health.
NOTES ON DIVERSITY IN LIVING ORGANISMS
Develop a thorough understanding of the basis of classification schemes proposed by various scientists.
Understand the utility of acceptance of Five-Kingdom Classification scheme proposed by R.H Whittaker along with its advantages and disadvantages.
Know the differences between the prokaryotic and eukaryotic cells and cellularity in living organisms.
Examples are very crucial in the study of diversity in the living world. Having the examples written in tabular forms of point-wise will help you in quick revision.
Refer to NCERT textbook for examples. Previous years&rsquo questions have shown that the examples have come directly from the NCERT text.
From the analysis of previous years&rsquo question papers, the animal kingdom becomes very important from the perspective of the number of questions.
Solve as many questions as possible. As the unit diversity in the living world gives a lot of factual information, it will be of great help that you prepare bullet notes from questions. This manner of reverse learning helps a lot with memorizing examples and facts.
Certain prokaryotes can live in extreme environments such as the Morning Glory pool, a hot spring in Yellowstone National Park. The spring’s vivid blue color is from the prokaryotes that thrive in its very hot waters. (credit: modification of work by Jon Sullivan)
In the recent past, scientists grouped living things into five kingdoms—animals, plants, fungi, protists, and prokaryotes—based on several criteria, such as the absence or presence of a nucleus and other membrane-bound organelles, the absence or presence of cell walls, multicellularity, and so on. In the late 20 th century, the pioneering work of Carl Woese and others compared sequences of small-subunit ribosomal RNA (SSU rRNA), which resulted in a more fundamental way to group organisms on Earth. Based on differences in the structure of cell membranes and in rRNA, Woese and his colleagues proposed that all life on Earth evolved along three lineages, called domains. The domain Bacteria comprises all organisms in the kingdom Bacteria, the domain Archaea comprises the rest of the prokaryotes, and the domain Eukarya comprises all eukaryotes—including organisms in the kingdoms Animalia, Plantae, Fungi, and Protista.
Two of the three domains—Bacteria and Archaea—are prokaryotic. Prokaryotes were the first inhabitants on Earth, appearing 3.5 to 3.8 billion years ago. These organisms are abundant and ubiquitous that is, they are present everywhere. In addition to inhabiting moderate environments, they are found in extreme conditions: from boiling springs to permanently frozen environments in Antarctica from salty environments like the Dead Sea to environments under tremendous pressure, such as the depths of the ocean and from areas without oxygen, such as a waste management plant, to radioactively contaminated regions, such as Chernobyl. Prokaryotes reside in the human digestive system and on the skin, are responsible for certain illnesses, and serve an important role in the preparation of many foods.
Metabolism of prokaryotic cells
Prokaryotic organisms have evolved a wide range of ways of to take energy from the environment. Compared to eukaryotic cells that have only evolved to transfer energy through photosynthesis and respiration, prokaryotic cells can obtain energy through photosynthesis, respiration, nitrogen fixation, denitrification, sulfate reduction and methanogenesis. These words may not mean anything to you but they illustrate the diversity of ways prokaryotic cells can take energy from their surrounding environment.
Introduction to Genome Biology and Diversity
Organisms display astonishing levels of cell and molecular diversity, including genome size, shape, and architecture. In this chapter, we review how the genome can be viewed as both a structural and an informational unit of biological diversity and explicitly define our intended meaning of genetic information. A brief overview of the characteristic features of bacterial, archaeal, and eukaryotic cell types and viruses sets the stage for a review of the differences in organization, size, and packaging strategies of their genomes. We include a detailed review of genetic elements found outside the primary chromosomal structures, as these provide insights into how genomes are sometimes viewed as incomplete informational entities. Lastly, we reassess the definition of the genome in light of recent advancements in our understanding of the diversity of genomic structures and the mechanisms by which genetic information is expressed within the cell. Collectively, these topics comprise a good introduction to genome biology for the newcomer to the field and provide a valuable reference for those developing new statistical or computation methods in genomics. This review also prepares the reader for anticipated transformations in thinking as the field of genome biology progresses.
Keywords: Chromatin DNA DNA replication Epigenetics Eukaryotes Gene structure Organelles Organism diversity Plasmids Prokaryotes Protein RNA Regulatory DNA Transcription Translation Viruses.
Archaeal surprises and uncultivable bacteria
Studies focusing on the Archaeal domain of life provide a continuous wealth of biological information that challenges old dogmas and preconceived ideas. Archaea have distinct molecular characteristics, some resembling Bacteria (e.g. genome organization, cellular structure) and others Eukarya (e.g. transcriptional machinery). Genomics revealed that the archaeal DNA replication machinery is a simplified version of the eukaryotic DNA replication apparatus. Stephen Bell (MRC Cambridge, UK) and Magnus Lundgren (Uppsala University, Sweden) provided an elegant example of the degree of similarity in DNA replication between archaeal and eukaryotic cells. They described independent studies revealing that Sulfolobus possesses multiple replication origins ( Lundgren et al., 2004 Robinson et al., 2004 ). Thus, these findings abolish the dogma that all prokaryotic chromosomes have a single origin of replication.
By using microarray-based marker frequency analysis, Lundgren showed that bidirectional replication is initiated in the Sulfolobus chromosome from three separate origins in near synchrony ( Lundgren et al., 2004 ). Furthermore, he showed that the replication forks of Sulfolobus advance at rates similar to those of eukaryotic replication forks (≈100 bp min −1 ) and much lower than E. coli elongation rates (≈1000 bp min −1 ). M. Lundgren also reported that, in contrast to initiation, replication termination in Sulfolobus occurred asynchronously with certain replication forks still progressing over 40 min after the others had terminated.
Bell described the high resolution in vitro and in vivo molecular characterization of two replication origins in Sulfolobus solfataricus using 2D gel analysis and replication initiation point mapping to reveal the precise initiation sites of bidirectional replication. He demonstrated that the three homologues in Sulfolobus of the eukaryotic initiator proteins Orc1 and Cdc6 have different specialized functions in vivo. DNA binding sites for Cdc6-like proteins exist in at least two of the replication origins of Sulfolobus. The cdc6-1 gene is located close to one origin of replication (oriC1), whereas cdc6-3 is located close to a second origin (oriC2). Bell demonstrated that different subsets of Cdc6 proteins bind to oriC1 and oriC2 in the G1 to S growth-phase transition. Cdc6-1 binds to oriC1, whereas both Cdc6-1 and Cdc6-3 bind to oriC2. Interestingly, Cdc6-2 binds to the origins of replication in G2, preventing Cdc6-1 and Cdc6-3 from binding and therefore providing a model for the regulation of origin activity ( Dionne et al., 2003 Robinson et al., 2004 ).
The most extremophilic cells on earth, from hyperthermophiles growing above 80°C, to acidophiles growing below pH 3, or halophiles growing at 3 M KCl are all Archaea. David Prangishvili (University of Regensburg, Germany) showed that phages from Archaea also rival common prokaryotic viruses in terms of resistance to harsh environments, diverse morphologies and extraordinary genome composition ( Rachel et al., 2002 Prangishvili and Garrett, 2004 ). In a systematic search for viruses in hot terrestrial environments of North America and Europe, Prangishvili and co-workers found 16 novel double-stranded DNA viruses from hyperthermophilic archaeal hosts (e.g. Sulfolobus, Acidianus, Thermoproteus, Pyrobaculum). Electron microscopic studies revealed particles with different morphotypes, from filamentous, rod-shaped and head-and-tail viruses to novel morphotypes previously not observed in nature, like balloon-shaped, ampulla-shaped and lemon-shaped viral particles. To classify these bizarre phages, three novel virus families have been introduced: Globuloviridae, Ampullaviridae and Bicaudaviridae. Surprisingly, some bicaudavirus particles are capable of undergoing a dramatic extracellular morphogenesis by extending two tails at the tips of their lemon-like capsids at temperatures above 75°C.
The morphology of these hyperthermophilic viruses is not their only astonishing feature. The sequencing of their genomes unveiled that most (>90%) of their putative genes, and sometimes all their open reading frames (ORFs), have no significant matches in current DNA databases, and are therefore considered as of unknown function. This fact suggests that these hyperthermophilic viruses have found completely novel molecular solutions for their biological functions. It is tempting to speculate whether these ‘novel’ (unknown for us) molecular apparatuses are kept in these viruses as molecular fossils from primitive cells of thermophilic character.
Equally astonishing is the identification of Nanoarchaeum equitans reported by Harald Huber (University of Regensburg, Germany) ( Huber et al., 2002 ). This extremely small microorganism (400 nm in diameter, representing 1% of the cell volume of E. coli) was isolated as an obligate symbiont/parasite from new species of the hyperthermophilic chemolithoautotrophic Archaea Ignicoccus (a organism with an inner and outer membrane and a large periplasmic-like space). Cells of N. equitans are spherical and grow attached to the surface of Ignicoccus cells growing under anaerobic conditions at temperatures between 75 and 98°C (Fig. 3). The genome of N. equitans is only 0.49 Mb and lacks most biosynthetic and metabolic pathways. For instance, lipids from its cell membrane derived from the Ignicoccus inner membrane. However, N. equitans has a hexagonal S-layer. N. equitans represents a novel phylum of Archaea, the Nanoarchaeota, with unique 16S RNA sequences. Using specific primers for the signature sequences found in the 16S RNA genes of N. equitans, Huber and co-workers found evidence of a worldwide distribution of Nanoarchaeota ( Hohn et al., 2002 Huber et al., 2003 ).
Transmission electron micrograph of three cells of Nanoarchaeum equitans attached on the surface of an Ignicoccus cell (right). Platinum shadowed. Bar: 1 µm. (This electron micrograph was kindly provided by Harald Huber, University of Regensburg, Germany.)
Apart from Nanoarchaeota, the two classical phyla of Archaea are the Crenarchaeota and Euryarchaeota ( Forterre et al., 2002 ). Indications of the existence of unculturable Archaea belonging to a third phylum, the Korarchaeota, have been obtained from environmental DNA sequences ( Barns et al., 1996 ). Using a similar approach, Christa Schleper (Darmstadt University of Technology, Germany) reported the detection of novel phylogenetic lineages of uncultivated Crenarchaeota in low to moderate temperature, marine and terrestrial environments. Using 16S rDNA-based phylogenetic surveys, Schleper and co-workers found one particular lineage of crenarchaeota in most soil and water samples ( Ochsenreiter et al., 2003 ). The use of 16S rDNA surveys has revealed that microbial diversity in soil is extremely high and current estimates suggest that > 99% of existing microorganisms have not been cultured or characterized. The Schleper lab has constructed complex large-insert metagenomic libraries from soil DNA to access the genomes of these uncultivable microorganisms, including the Crenarchaeota and Acidobacteria, a novel phylum of uncultivable Bacteria that is detected (by 16S rDNA screening) in many different habitats around the globe ( Quaiser et al., 2002 2003 ).
Translation in Prokaryotes | Genetics
The process by which proteins are produced with amino acid sequences specified by the sequence of codons in messenger RNA is called translation. Translation is the first stage of protein biosynthesis.
The main points about translation in prokaryotes are given below:
Translation occurs in the cytoplasm where the ribosomes are located. Ribosomes are made of a small and large subunit which surrounds the mRNA. In prokaryotic translation 70S ribosomes with 30S and 50S subunits are used. The mRNA is synthesized from DNA only. In prokaryotes, there are several initiation and termination sites.
In translation, messenger RNA (mRNA) is decoded to produce a specific polypeptide according to the rules specified by the genetic code. This uses an mRNA sequence as a template to guide the synthesis of a chain of amino acids that form a protein. Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA are not necessarily translated into an amino acid sequence.
The translation process requires mRNA, rRNA, ribosomes, 20 kinds of amino acids and their specific tRNAs.
In prokaryotes, three factors are involved in the initiation of translation [IF 1, IF 2 and IF 3], one factor in the elongation of polypeptide chain and three factors in chain termination [RF1, RF2 and RF3],
Two types of enzymes are used in translation. Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding between specific tRNAs and the amino acids. The enzyme peptidyl transferase connects A site and P site by forming a peptide bond [the nitrogen carbon bond] during elongation phase.
In the process of translation two types of codons, viz., start codon and stop codons are involved. The codon, AUG, initiates the process of translation and one of three stop codons i.e. UAA, UAG or UGA is used for chain termination.
7. Starting Amino Acid:
In prokaryotes, starting amino acid is N-formyl methionine. Moreover, there is overlapping of transcription and translation.
Mechanism of Translation in Prokaryotes:
Translation process consists of three major phases or stages, viz:
These are briefly discussed below:
This is the first phase of translation. Start or initiation codon [AUG] is responsible for initiation of translation process.
Initiation of translation in prokaryotes involves the assembly of the components of the translation system which are: the two ribosomal subunits (small and large), the mRNA to be translated, the first (formyl) aminoacyl tRNA (the tRNA charged with the first amino acid), GTP (as a source of energy), and three initiation factors (IF 1, IF 2 and IF 3) which help the assembly of the initiation complex.
The ribosome consists of three sites, the A site, the P site, and the E site. The A site is the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fMet-tRNAf Met , which enters at the P site). The P site is where the peptidyl tRNA is formed in the ribosome. And the E site which is the exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide chain.
Translation begins with the binding of the small ribosomal subunit to a specific sequence on the mRNA chain. Initiation of translation begins with the 50S and 30S ribosomal subunits. IF1 (initiation factor 1) blocks the A site to ensure that the IMet-tRNA can bind only to the P site and that no other aminoacyl-tRNA can bind in the A site during initiation, while IF3 blocks the E site and prevents the two subunits from associating.
IF2 is a small GTPase which binds fmet-tRNAf Met and helps its binding with the small ribosomal subunit. The 3′ end of the 16S rRNA of the small 30S ribosomal subunit recognizes the ribosomal binding site on the mRNA (Shine-Dalgarno sequence or SD), through its anti-SD sequence, 5-10 base pairs upstream of the start codon. The Shine-Dalgarno sequence is found only in prokaryotes.
This helps to correctly position the ribosome onto the mRNA so that the P site is directly on the AUG initiation codon. IF3 helps to position fMet-tRNAf met into the P site, such that fMet-tRNAf met interacts via base pairing with the mRNA initiation codon (AUG). Initiation ends as the large ribosomal subunit joins the complex causing the dissociation of initiation factors.
The small subunit binds via complementary base pairing between one of its internal subunits and the ribosome binding site. This site a sequence of about ten nucleotides on the mRNA. It is located anywhere from 5 and 11 nucleotides from the initiating codon [AUG],
After binding of the small subunit, a special tRNA molecule, called N-formyl methionine, or fMet, recognizes and binds to the initiator codon. Then the large subunit binds resulting in the formation of the initiation complex. As soon as the initiation complex is formed, the fMet-tRNA occupies the P site of the ribosome and the A site is left empty.
This entire initiation process is facilitated by extra proteins, called initiation factors that help with the binding of ribosomal subunits and tRNA to the mRNA chain.
This is the second phase or middle phase of translation. Elongation begins after the formation of the initiation complex. Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit.
Elongation starts when the fmet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor-T4 (EF-T4), a small GTPase. Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next amino acid to be added to the peptide chain.
The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site.
This process, known as peptide bond formation, is catalyzed by a ribozyme, peptidyltransferase, an activity intrinsic to the 23S ribosomal RNA in the 50S ribosomal subunit. Now, the A site has newly formed peptide, while the P site has an unloaded tRNA (tRNA with no amino acids). In the final stage of elongation, translocation, the ribosome moves 3 nucleotides towards the 3′ end of mRNA.
Since tRNAs are linked to mRNA by codon-anticodon base-pairing, tRNAs move relative to the ribosome taking the nascent polypeptide from the A site to the P site and moving the uncharged tRNA to the E exit site.
This process is catalyzed by elongation factor G (EF-G). The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA binds to the A site, until the ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).
When the A site opens again, the next appropriate aminoacyl tRNA can bind there and the same reaction takes place, yielding a three-amino acid peptide chain. This process repeats, creating a polypeptide chain in the P site of the ribosome. A single ribosome can translate 60 nucleotides per second. This speed can be vastly augmented when ribosomes unite together to form polyribosomes.
This is the last phase of translation. Termination occurs when one of the three termination codons moves into the A site. These codons are not recognized by any tRNAs. Instead, they are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons).
These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome. A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process.
DNA Replication in Prokaryotes
The prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and highly coiled around proteins. While there are many similarities in the DNA replication process, these structural differences necessitate some differences in the DNA replication process in these two life forms. DNA replication in prokaryotes has been extensively studied, so we will learn the basic process of prokaryotic DNA replication, then focus on the differences between prokaryotes and eukaryotes.
How does the replication machinery know where to start? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. E. coli has a single origin of replication on its one chromosome, as do most prokaryotes (Figure 1). The origin of replication is approximately 245 base pairs long and is rich in AT sequences. This sequence of base pairs is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process because it requires energy. As the DNA opens up, Y-shaped structures called replication forks are formed (Figure 1). Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins (Figure 2) coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix.
Figure 1: DNA replication in prokaryotes, which have one circular chromosome.
The next important enzyme is DNA polymerase III, also known as DNA pol III, which adds nucleotides one by one to the growing DNA chain (Figure 2). The addition of nucleotides requires energy this energy is obtained from the nucleotides that have three phosphates attached to them. ATP structurally is an adenine nucleotide which has three phosphate groups attached breaking off the third phosphate releases energy. In addition to ATP, there are also TTP, CTP, and GTP. Each of these is made up of the corresponding nucleotide with three phosphates attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the existing chain.
In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for DNA synthesis DNA pol I is used later in the process and DNA pol II is used primarily required for repair (this is another irritating example of naming that was done based on the order of discovery rather than an order that makes sense).
DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction (a new DNA strand can be only extended in this direction). It requires a free 3′-OH group (located on the sugar) to which it can add the next nucleotide by forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3′-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3′-OH end. Another enzyme, RNA primase, synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA. RNA primase does not require a free 3′-OH group. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand (Figure 2).
Figure 2 A replication fork is formed when helicase separates the DNA strands at the origin of replication. The DNA tends to become more highly coiled ahead of the replication fork. Topoisomerase breaks and reforms DNA’s phosphate backbone ahead of the replication fork, thereby relieving the pressure that results from this supercoiling. Single-strand binding proteins bind to the single-stranded DNA to prevent the helix from re-forming. Primase synthesizes an RNA primer. DNA polymerase III uses this primer to synthesize the daughter DNA strand. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in short stretches called Okazaki fragments. DNA polymerase I replaces the RNA primer with DNA. DNA ligase seals the gaps between the Okazaki fragments, joining the fragments into a single DNA molecule. (credit: modification of work by Mariana Ruiz Villareal)
The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only extend in the 5′ to 3′ direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel that is, one strand is in the 5′ to 3′ direction and the other is oriented in the 3′ to 5′ direction. One strand, which is complementary to the 3′ to 5′ parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5′ to 3′ parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand.
The leading strand can be extended by one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3′ to 5′, and that of the leading strand 5′ to 3′. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up it does so by causing temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA pol I, which breaks down the RNA and fills the gaps with DNA nucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment.
(Lisa’s note: I think this process is almost impossible to visualize from reading text. I strongly recommend that you watch a couple of animations / videos like the one available here. There are additional links in Blackboard)
Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division. The process of DNA replication can be summarized as follows:
- DNA unwinds at the origin of replication.
- Helicase opens up the DNA-forming replication forks these are extended in both directions.
- Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
- Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling (over-winding).
- Primase synthesizes RNA primers complementary to the DNA strand.
- DNA polymerase III starts adding nucleotides to the 3′-OH (sugar) end of the primer.
- Elongation of both the lagging and the leading strand continues.
- RNA primers are removed and gaps are filled with DNA by DNA pol I.
- The gaps between the DNA fragments are sealed by DNA ligase.
Table 1: The enzymes involved in prokaryotic DNA replication and the functions of each.
|Prokaryotic DNA Replication: Enzymes and Their Function|
|DNA pol I||Exonuclease activity removes RNA primer and replaces with newly synthesized DNA|
|DNA pol II||Repair function|
|DNA pol III||Main enzyme that adds nucleotides in the 5′-3′ direction|
|Helicase||Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases|
|Ligase||Seals the gaps between the Okazaki fragments to create one continuous DNA strand|
|Primase||Synthesizes RNA primers needed to start replication|
|Sliding Clamp||Helps to hold the DNA polymerase in place when nucleotides are being added|
|Topoisomerase||Helps relieve the stress on DNA when unwinding by causing breaks and then resealing the DNA|
|Single-strand binding proteins (SSB)||Binds to single-stranded DNA to avoid DNA rewinding back.|
DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome and large number of variants available. Escherichia coli has 4.6 million base pairs in a single circular chromosome, and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the chromosome in both directions. This means that approximately 1000 nucleotides are added per second. The process is much more rapid than in eukaryotes.


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domain: artistic


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- .Lv 77 years agoFavorite Answer
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domain: medical


text:
This paper presents the impact of selected essential aspects of geological structure on the state of slope stability as assessed by changes in the safety factor (SF). The engineering geological slope features that were analysed include: (i) changes in soil cohesion and internal friction angle, (ii) presence of a weak soil layer and its span, (iii) structural discontinuity (i.e. fault), (iv) soil permeability properties and water conditions (i.e. groundwater level, long and heavy rainfall). A certain scheme of slope stability calculations has been proposed for a case involving complex geological conditions. The numerical modelling of a hypothetic slope was performed using the shear strength reduction method based on the finite element method.
Stability of slopes is a topical and substantial issue, affecting areas where there are natural slopes as well as man-made slopes in urban areas, e.g. railway and road embankments, deep excavations or dams [1, 2, 3]. In these all cases, there are a large number of factors influencing the final safety factor value. Furthermore, numerical evaluation of the slope equilibrium state requires accurate representation in a computational model of soil and water conditions, which are determined by the geological structure. This is particularly important in areas which have been tectonically active or subject to intensive geodynamic processes in their geological history [e.g. 4, 5, 6, 7]. This is due to the potential occurrence of peculiar geological features, such as soil layers with reduced strength parameters [e.g. 8], layering associated with strength anisotropy [e.g. 9] and the presence of fault or discontinuity zones in the soil or rock mass [e.g. 10, 11]. For this reason, numerical models often do not adequately replicate real geological conditions. As a consequence, the modelled equilibrium state may not accurately correspond to reality and the computational geometry of the mass movement slip surface may be incorrect.
Moreover, in numerical modelling there are various consequences deriving from the calculation method selected for slope stability analysis, as discussed in  and . Appropriate definition of the slope numerical model is also important (including: right type, sufficient number and proper size of finite elements). These aspects of modelling were discussed by the authors in an earlier publication .
This paper presents universal multistep significance analyses of the impact of individual specific soil and water conditions of a theoretical slope on its stability, expressed in terms of safety factor (SF). An evaluation of this type should be conducted in each case where there is a complex geological structure, on the basis of a properly planned parametric analysis, taking into account different cases of selected geological features.
2 Materials and methods
The shear strength reduction method (so called c-ϕ reduction method) was used for the purpose of slope stability analysis. This method was implemented in Z_soil v16.08 software based on the finite element method (FEM), as described in detail by among others [14, 15].
The analysed slope was 25 m high and the slope angle α was 30∘. In the reference case, the slope was made of homogeneous material with the following parameters: γ = 22 kN/m3, c = 10 kPa, ϕ = 30∘. This soil was modelled using the Mohr-Coulomb (elastic-perfectly plastic) constitutive model. In addition, two other materials were used in the analysis. The first material was an isotropic soil with strength parameters reduced by one-third compared to the reference case. The second material was transversally isotropic (i.e. multilaminate) in order to simulate anisotropic material behaviour. In this case, a matrix of material parameters (as in the previous case) and weakness planes strength parameters (reduced by one-third) were defined. The weakness planes dips were consistent and equal to the slope inclination. In the numerical calculations non-associated flow law was used for elastic-plastic constitutive models.
Figure 1 shows the scheme of subsequent stages of the numerical analysis performed for selected components of soil and water conditions. The presented workflow is dedicated for near-surface landslides cases (related to e.g. surface soils or rock debris), opposite to very deep and complex models driven by regional geotectonic regimes.
Study of the real case according to the proposed scheme can show the scale, complexity and scope of the problem which is analysed. For real conditions, some of the factors analysed may be of little importance,while others may be taken into account in various ways. Therefore, the proposed scheme contains criteria that can be used to determine whether or not a given factor should be taken into account.
It should also be emphasised that the data needed to carry out the proposed analyses are not deterministic but they vary in relation to regional geological engineering conditions. Similarly, all analysed structural factors (e.g. thickness and location of the "weak layer", size and orientation of discontinuities, strength parameters of the soil/rock mass and contact interface, etc.) for some regions will be random to some extent. This randomness range as well as geological features recognitions defines the limits of parameter values for the sensitivity analysis.
In stage one, the impact of variation in strength parameters on SF were analysed. The values of these parameters are characterized by limited certainty, which may be attributed to soil variability as well as errors in the underlying tests. First, cohesion was incrementally changed at a constant value of the friction angle of soil. After each change of cohesion, the SF value was recalculated. Next, in a similar manner, the angle of friction was incrementally reduced at a constant value of cohesion. Based on the relations SF(c) and SF(ϕ), a triaxial graph of SF as a function of cohesion and internal friction angle of soil was developed. These correlations were established as a surface, which was subsequently approximated by a plane in order to determine an empirical formula for the safety factor.
In stage two, a multi-case analysis was conducted regarding the impact of the presence of a weak soil layer at different depths below terrain level. This analysis made it possible to identify the worst case scenario. Based on that (i.e. the worst case in a plane strain analysis), the study was continued with a three dimensional models analysis, so as to determine the impact of the span of a layer of reduced shear strength on the slope equilibrium state. Figure 2 shows two example models (which may represent e.g. tills, clayey sands highland or weathered colluvium - clayey soil - on the slope) with different weak layer widths.
Sedimentation and diagenesis processes may result in rock structure anisotropy. Likewise weathering which can also affect the soil structure. Macroscopic manifestations of such anisotropy may include layering, lamination or a system of fractures (see Figure 3), translating into soil strength anisotropy. In the case of such materials, the use of isotropic soil models is undermined by potential major errors. Such models assume that soil strength is not dependent on the angle of load application. However, inter alia  provides an example of Tournemire shale to demonstrate the strength anisotropy effect: during triaxial shear tests, the strength of this material at uniaxial compression parallel to layering may be reduced by 30 to 50% compared to strength at uniaxial compression perpendicular to layering.
In modelling strength reduction (by one third in terms of both cohesion and internal friction angle), i.e. strength anisotropy of slope formations, two different approaches (A and B) were followed and a comparison of their outcomes was made. Approach A involved using interface elements in a numerical model, as they enable manual modification of strength parameters on the relevant surfaces defined by lines (see Figure 4A). These surfaces, which may be curvilinear, are input separately. Thanks to this approach it is possible to introduce even complete material discontinuity. In the alternative approach B, we replicated anisotropy with a multilaminate constitutive model. The model belongs to a group of elastic-ideally plastic constitutive models, and it is dedicated to layered materials, such as shale . In this model, it is assumed that there are linear weakness planes in a geomaterial, parallel to one another, along which displacement occurs (see Figure 4B). Weakness planes can be determined by systems with different angles relative to each other. These systems will be present throughout the defined material. On these weakness planes, the maximum strength is limited by the Mohr-Coulomb failure criterion. Various strength parameters can be determined for each of the weakness planes systems. Nevertheless, it is not possible to define strength parameters of individual weakness surfaces.
Another element of a geological structure that was analysed was structural discontinuity in the form of a fault at the base of the slope. Such elements are often neglected in numerical model construction due to insufficient depth of geological structure investigation. In the considered cases, the location of the fault was assumed under the slope, in order to verify the interactions with near-surface landslides (i.e. the possibility of extending the slip surface). As in the case of strength anisotropy modelling, we followed two approaches (A and B). They were similar to the ones used in earlier calculations (for shear strength reduction by one third). In this case only one interface-type surface was used in approach A, while approach B involved a limited discontinuity zone wherematerial was defined with a multilaminate model (with weakness planes dipping at the same angle as the fault and slope inclination; see Figure 5). The fault zone is defined as multilaminate material in order to model zone filled with fractured material. An example of such use of a multilaminate model can be found in a paper by , who presented a case study of tunnel drilling in a faulted rock mass.
Numerical analyses of slope stability among others provide quantity evaluation of water level impact on equilibrium state [e.g. 21] as well as pore water distribution [e.g. 22]. In the presented study, the analysed scenarios focused on rainfall. This was further to analysis of 74 landslides in the Carpathian flysch belt activated in 2010, which indicated rainwater infiltration as one of the three main factors triggering landslide movements and the cause of one in five landslides .Water pressure plays a major role in destabilizing slopes, as has been demonstrated multiple times in wide variety of cases [e.g. 21, 24, 25]. In order to analyse potential changes in the slope equilibrium state resulting from heavy rainfall, a model of a slope made of a homogeneous material (as described above) of the filtration coefficient k = 10−5 m/s (see Figure 6A) were developed. For the purpose of the simulation, we assumed an infiltration coefficient of 0.3, presence of groundwater as well as rainfall of growing intensity, producing an effect of water flow (expressed in m3/d) to the slope surface (as shown in Figure 6B). In other words, rainfall has been simulated as an inflow boundary condition (as suggested by ), where value of inflow varies in time.
3 Results, interpretations and discussion
In the reference case, the slope safety factor was 1.35. Figure 7A shows the geometry of the relevant slip surface. In the subsequent stage, in line with the procedure described above, the cohesion and internal friction angle were incrementally changed. Based on the results of a series of SF calculations, the relation shown in Figure 7B was determined. This formula enables quick evaluation of the trend of changes and the stability safety margin for various intervals of cohesion and internal friction angle. Due to the practical application of the formula, it was decided to use the mathematical relation with friction angle values in degrees.
For slopes of simple geological structure, quasi-linear dependence of SF on strength parameters enables extrapolation of results for new values of c and ϕ. However, when analysing slopes of more complex structure, the SF coefficient of determination (R2) needs to be considered to evaluate the usefulness of the outcome formula. In the analysed case, the slope equilibrium state is more sensitive to a reduction in internal friction angle, which stems from a homogenous structure and the Mohr-Coulomb criterion. A decrease in friction angle by 9∘ (at c = 10 kPa) results in a loss of stability, whereas a reduction of cohesion to 0 (at ϕ = 30∘) does not result in a loss of stability (due to the angle of slope).
After determining the impact of variation in strength parameters of the material forming the entire slope, an analysis was carried out of the impact of the presence of a 5 m layer of reduced shear strength (one third weaker than the reference material; see Figure 8). As the depth of the weaker soil layer roof increased, the value of SF decreased; in the worst case, SF was reduced by approximately 30%. The critical position of the new layer was at the bottom of the slope and 20 m below terrain level. Such an outcome is related to the presence of the longest section of the slip surface within the weaker layer. In order to analyse the impact of the span of a weaker layer on the slope, a three dimensional numerical model based on the worst case in a plane strain analysis (PSA) was created (see Figure 9).
At the further stage, the stability analysis clearly demonstrates that the impact of a reduced strength parameters layer decreases, as its width decreases, which is related to the strengthening of the lateral zones of soil. However, if a certain width is exceeded, the slope equilibrium state becomes similar to the stability of a specific case in PSA (see Figure 9) which assumes ‘infinite’ width of both the layer and the slope. Based on the study results, it can be concluded that three dimensional modelling provides optimisation of slope protection costs, as was also noted by . Figure 9 also shows the geometry of the slip surface in a three dimensional displacement analysis, which is derived from the plain strain analysis outcome.
In the next two models of the analysed slope, strength anisotropy through interface elements and a multilaminate model, respectively, were introduced. The results of an analysis for these two approaches differ significantly (see Figure 10). In both models, there wasa major decrease in SF; in the one with interface elements, SF even fell below the critical value of 1. Moreover, displacement fields resulting from loss of stability are also different. Interface elements enabled replication of several failure surfaces, while the multilaminate model enabled development of a translational landslide. Presumably, the reason why the results of the SF calculations in the first approach are lower is that after loss of stability displacements can develop in an unlimited manner, whereas in the second approach displacements encounter soil zones which have not been plasticised. Based on such different, yet characteristic, effects, it is possible to select a model that will best correspond to the actual soil displacements observed. Most likely, for slopes made of stiff formations with high strength reduction depending on orientation (e.g. shale), the process of loss of stability will resemble the one modelled in the first approach.
The weakness planes modelling methods described above were also used to simulate a slope with a structural discontinuity surface at the base. In this case, the analysis outcome, SF=1.2, was the same for both approaches. However, displacement fields were still different. This time, in the first approach the displacement field is related to surficial translational slides, whereas when a multilaminate model is used in the fault zone, the entire soil mass is subject to landslide movements. Whenever areas affected by tectonic movements (such as the hills in the Carpathian flysch region) are modelled, it is frequently debated whether a surficial slide or a landslide with a deeply located displacement surface/zone occurs within a slope. The selection of the right approach to the modelling of particular slopes may be based on deep investigation of their geological structure and the results of inclinometer measurements.
The geological structure of numerous slopes, together with their geometry and groundwater level, does not present a risk of activation of landslide processes. However, a factor which can drastically change the equilibrium state is soil/rock filtration properties combined with long and intensive rainfall. An example of this situation was modelled in the last stage of this numerical analysis. Figure 12 presents gradual changes in the degree of soil saturation of a slope as a result of continuous rainfall until the moment when the slope equilibrium state reaches the critical value SF=1. As a consequence, landslide movements are activated in a slope which was originally stable (SF=1.35).
The issue of convenient guidance for numerical analysis of slope stability in the presented paper is important because of the need for widespread use. New challenges like this which are related to anthropression (e.g. development of areas of man-made soils; [28, 29] or which become apparent due to new slope stability evaluation numerical methods [30, 31] require ongoing discussion in search of optimal solutions that can be quickly applied in the various cases along with scientific research and engineering practice .
At the beginning of the discussion, much attention should be paid to the calculation method. The c-ϕ reduction method is also implemented in software other than Z_soil, which also use FEM or methods such as distinct element method and finite difference method (e.g. Plaxis, FLAC, UDEC, PFC, Midas, GEO5 MES). A discusion about calculation methods does not lie at the heart of this paper, but is merely indicated as one of the aspects which might affect the SF results. In some particularly complex cases, calculations by two methods or even more may usefully be carried out, followed by a comparative approach to the differences. Moreover, the results of calculations using different methods can be used at various stages of geological structure recognition (e.g. initial slope stability calculations, stability calculations of individual smaller slope parts, stability calculations of the entire slope model). For instance, such approach can be found, in the article on numerical modeling of the deep mining in Sweden . Furthermore, in some cases of slope stability analysis (e.g. slopes in simple geological conditions) analytical methods, too, provide correct SF results, as has been shown in many papers, e.g. .
Another aspect of numerical calculations are material models. The numerical calculations in the presented study employ basic and not overly complicated constitutive material models. For some particular soils and in some sophisticated studies there is a need for more advanced models (as introduced in [35, 36]). Nevertheless, their use requires more uncommon soil parameters.
In some unty pical cases, the proposed calculation scheme will serve as a useful starting point for introducing modifications. Such a situation may occur, for example in areas that are strongly impacted by human endeavour, by e.g. highway construction, suburban development, or building of industrial sewage pipes . The issue of selecting appropriate destabilizing factors lies at the heart of the basic framework for landslide risk assessment and management (described in ). Another example is a field characterized by highly developed vegetation, conditioning reinforcement or shallow surface landslides [39, 40, 41]. Furthermore, the geotechnical reinforcment of a slope with a weak layer (in 3D FEM approach) can be considered a component of the calculation scheme [42, 43].
Through taking a factor-by-factor approach, the impact that individual components of key slope features have on stability can be quantified (observed as changes in SF value). Based on these outcomes, their equivalents can be established, as was presented in . In the real case, a positive development of the presented method is the additional calculations of stability, with all characteristics of the analysed slope for cumulative effect. Then, an observational method can be used to assess the reliability of the final results. Verification of this type is applied in various aspects of numerical calculations of the soil medium, such as subsidence or soil bearing capacity [45, 46, 47] which are also linked with the assessment of slope stability. The differences found in numerical calculations and field observations can be used to calibrate the numerical model .
Geophysical methods are one of the most frequently proposed methods for investigating the geological structure and so is proposed in the presented calculation scheme. However, one should remain prudent when using these tools, as they have their own flaws "imperfections". For instance,  indicate the possibility that weak and small layers are overlooked in the ERI method. In the case of slopes, such layers maybe responsible for the emergence of predisposed slip surfaces, as presented in the current paper or in [27, 42]. Dahlin and Zhou  introduce comparison of 2D resistivity imaging electrode arrays, with their vertical and horizontal resolution. To enhance the certainty of geophysical prospecting interpretations it is useful to perform several different geophysical field tests, due to their various advantages and disadvantages [51, 52].
Numerical analyses are widely-used tools for recognition of stress and displacements fields which deepen knowledge of the mechanisms involved in landslides. Numerical models based on suitable in situ and laboratory tests calibrated with field observations, measurements of inclinometers, piezometric water level and land surveying make it possible to reliably predict changes in slope stability and in the event of an impending risk of a catastrophic landslide they provide the scientific support needed to trigger slope reinforcement works.
A multi-case numerical analysis can be used to identify the scope of outcomes along with the sensitivity of the model to variation in its different components (including erroneous assumptions made for the purpose of analysis).
The proposed scope of analysis of the slope model’s usefulness:
Mesh influence analysis (i.e. analysis of the geometry, number and size of finite elements, etc.);
Analysis of the model’s sensitivity to variation in strength parameters;
Comparison of the outcomes of a plane strain analysis and a three dimensional analysis;
The impact of peculiar soil features (e.g. strength anisotropy or soil layers of reduced shear strength) and structural discontinuities (e.g. faults) on the slope equilibrium state;
Study of changes in groundwater levels caused by extreme weather (e.g. heavy rainfall, increased levels of water reservoirs and rivers).
For real conditions, some of the analysed factors (depending from geological engineering conditions and it’s recognition) may be of little importance, while others may be taken into account in various ways. Therefore, the proposed scheme (at the Figure 4) contains criteria that decide whether or not to take into account a given factor and how to model it.
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domain: geography