General Microbiology Textbook PDF

General Microbiology General Microbiology LINDA BRUSLIND OREGON STATE UNIVERSITY CORVALLIS, OR General Microbiology Copyright © 2019 by Linda Bruslind is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted. Publication and ongoing maintenance of this textbook is possible due to grant support from Oregon State University Ecampus. Suggest a correction (bit.ly/33cz3Q1) Privacy (open.oregonstate.education/privacy) Contents 1. Introduction to Microbiology 1 2. Microscopes 7 3. Cell Structure 24 4. Bacteria: Cell Walls 31 5. Bacteria: Internal Components 37 6. Bacteria: Surface Structures 45 7. Archaea 52 8. Introduction to Viruses 59 9. Microbial Growth 67 10. Environmental Factors 72 11. Microbial Nutrition 79 12. Energetics & Redox Reactions 86 13. Chemoorganotrophy 93 14. Chemolithotrophy & Nitrogen Metabolism 100 15. Phototrophy 107 16. Taxonomy & Evolution 116 17. Microbial Genetics 122 18. Genetic Engineering 129 19. Genomics 138 20. Microbial Symbioses 144 21. Bacterial Pathogenicity 150 22. The Viruses 157 Creative Commons License 167 Recommended Citations 168 Versioning 170 1. Introduction to Microbiology Welcome to the wonderful world of microbiology! Yay! So. What is microbiology? If we break the word down it translates to “the study of small life,” where the small life refers to microorganisms or microbes. But who are the microbes? And how small are they? Generally microbes can be divided into two categories: the cellular microbes (or organisms) and the acellular microbes (or agents). In the cellular camp we have the bacteria, the archaea, the fungi, and the protists (a bit of a grab bag composed of algae, protozoa, slime molds, and water molds). Cellular microbes can be either unicellular, where one cell is the entire organism, or multicellular, where hundreds, thousands or even billions of cells can make up the entire organism. In the acellular camp we have the viruses and other infectious agents, such as prions and viroids. In this textbook the focus will be on the bacteria and archaea (traditionally known as the “prokaryotes,”) and the viruses and other acellular agents. Characteristics of Microbes Obviously microbes are small. The traditional definition describes microbes as organisms or agents that are invisible to the naked eye, indicating that one needs assistance in order to see them. That assistance is typically in the form of a microscope of some type. The only problem with that definition is that there are microbes that you can see without a microscope. Not well, but you can see them. It would be easy to dismiss these organisms as non-microbes, but in all other respects they look/act/perform like other well-studied microbes (who follow the size restriction). So, the traditional definition is modified to describe microbes as fairly simple agents/organisms that are not highly differentiated, meaning even the multicellular microbes are composed of cells that can act independently– there is no set division of labor. If you take a giant fungus and chop half the cells off, the remaining cells will continue to function unimpeded. Versus if you chopped half my cells off, well, that would be a problem. Multicellular microbes, even if composed of billions of cells, are relatively simple in design, usually composed of branching filaments. It is also acknowledged that research in the field of microbiology will require certain common techniques, largely related to the size of the quarry. Because microbes are so small and there are so many around, it is important to be able to isolate the one type that you are interested in. This involves methods of sterilization, to prevent unwanted contamination, and observation, to confirm that you have fully isolated the microbe that you want to study. Microbe Size Since size is a bit of theme in microbiology, let us talk about actual measurements. How small is small? The cellular microbes are typically measured in micrometers (µm). A typical bacterial cell (let us say E. coli) is Introduction to Microbiology | 1 about 1 µm wide by 4 µm long. A typical protozoal cell (let us say Paramecium) is about 25 µm wide by 100 µm long. There are 1000 µm in every millimeter, so that shows why it is difficult to see most microbes without assistance. (An exception would be a multicellular microbe, such as a fungus. If you get enough cells together in one place, you can definitely see them without a microscope!) When we talk about the acellular microbes we have to use an entirely different scale. A typical virus (let us say influenza virus) has a diameter of about 100 nanometers (nm). There are 1000 nanometers in every micrometer, so that shows why you need a more powerful microscope to see a virus. If a typical bacterium (let us pick on E.coli again) were inflated to be the size of the Statue of Liberty, a typical virus (again, influenza virus works) would be the size of an adult human, if we keep the correct proportions. http://learn.genetics.utah.edu/content/cells/scale/ The Discovery of Microbes The small size of microbes definitely hindered their discovery. It is hard to get people to believe that their skin is covered with billions of small creatures, if you cannot show it to them. “Seeing is believing,” that is what I always say. Or someone says that. In microbiology, there are two people that are given the credit for the discovery of microbes. Or at least providing the proof of their discovery, both around the same time period: Robert Hooke (1635-1703) Robert Hooke was a scientist who used a compound microscope, or microscope with two lenses in tandem, to observe many different objects. He made detailed drawings of his observations, publishing them in the scientific literature of the day, and is credited with publishing the first drawings of microorganisms. In 1665 he published a book by the name of Micrographia, with drawing of microbes such as fungi, as well as other organisms and cell structures. His microscopes were restricted in their resolution, or clarity, which appeared to limit what microbes he was able to observe. Antony van Leeuwenhoek (1632-1723) Antony van Leeuwenhoek was a Dutch cloth merchant, who also happened to dabble in microscopes. He constructed a simple microscope (which has a single lens), where the lens was held between two silver plates. Apparently he relished viewing microbes from many different sample types – pond water, fecal material, teeth scrapings, etc. He made detailed drawings and notes about his observations and discoveries, sending them off to the Royal Society of London, the scientific organization of that time. This invaluable record clearly indicates that he saw both bacteria and a wide variety of protists. Some microbiologists refer to van Leeuwenhoek as the “Father of Microbiology,” because of his contributions to the field. 2 | Introduction to Microbiology Microbial Groups Classification of organisms, or the determination of how to group them, continually changes as we acquire new information and new tools of assessing the characteristics of an organism. Currently all organisms are grouped into one of three categories or domains: Bacteria, Archaea, and Eukarya. The Three Domain Classification, first proposed by Carl Woese in the 1970s, is based on ribosomal RNA (rRNA) sequences and widely accepted by scientists today as the most accurate current portrayal of organism relatedness. Tree of Life. Introduction to Microbiology | 3 Bacteria The Bacteria domain contains some of the best known microbial examples (E. coli, anyone?). Most of the members are unicellular (but not all!), most members lack a nucleus or any other organelle, most members have a cell wall with a particular substance known as peptidoglycan (not found anywhere else but in bacteria!), they have 70S ribosomes, and humans are intimately familiar with many members, since they are common in soil, water, our foods, and our own bodies. All Bacteria are considered microbes. Archaea Archaea is a relatively new domain, since these organisms used to be grouped with the bacteria. There are some obvious similarities, since they are mostly (but not all!) unicellular, cells lack a nucleus or any other organelle, they have 70S ribosomes, and all Archaea are microbes. But they have completely different cell walls that can vary markedly in composition (but notably lack peptidoglycan and might have pseudomurien instead). In addition, their rRNA sequences have shown that they are not closely related to Bacteria at all. Eukarya The Eukarya Domain includes many non-microbes, such as animals and plants, but there are numerous microbial examples as well, such as fungi, protists, slime molds, and water molds. The eukaryotic cell type has a nucleus, as well as many organelles, such as mitochondria or an endoplasmic reticulum. They have 80S ribosomes and are commonly found as unicellular or multicellular. Viruses Viruses are not part of the Three Domain Classification, since they lack ribosomes and therefore lack rRNA sequences for comparison. They are classified separately, using characteristics specific to viruses. Viruses are typically described as “obligate intracellular parasites,” a reference to their strict requirement for a host cell in order to replicate or increase in number. These acellular entities are often agents of disease, a result of their cell invasion. Taxonomic Ranks Taxonomic ranks are a way for scientists to organize information about organisms, by determining relatedness. Domains are the largest grouping used, followed by numerous smaller groupings, where each 4 | Introduction to Microbiology smaller grouping consists of organisms that share specific features in common. Each level becomes more and more restrictive as to whom can be a member. Eventually we get down to genus and species, the groupings used for formation of a scientific name. This is the binomial nomenclature devised by Carl Linnaeus in the 1750s. Taxonomic Ranks. By Annina Breen (Own work) [CC BY-SA 4.0], via Wikimedia Commons Binomial Nomenclature When referring to the actual scientific name assigned to an organism, it is important to follow convention, so it is clear to everyone that you are referring to the scientific name. There are rules in science (just like in English class, where you would never refer to “mr. robert louis stevenson,” or at least not without expecting to get your paper back with red all over it). A scientific name is composed of a genus and a species, where the genus is a generic name and the species is specific. The species name, once assigned, is permanent for the organism, while the genus can change if new information becomes available. For example, the bacterium previously known as Streptococcus faecalis is now Enterococcus faecalis because sequencing information indicates that it is more closely related to the Introduction to Microbiology | 5 members of the Enterococcus genus. It is important to note that it is inappropriate to refer to an organism by the species alone (i.e. you should never refer to E. coli as “coli” alone. Other bacteria can have the species “coli” as well.) Now for the rules: The genus is always capitalized. The species is always lowercase. And both the genus and the species are italicized (common if typewritten) or underlined (common if handwritten). The genus may be shortened to its starting letter, but only if the name has been referred to in the text in its entirety at least once first (the exception to this is E. coli, due to its commonality, where hardly anyone spells out the Escherichia genus anymore). Key Words microbiology, microorganisms, microbes, unicellular, multicellular, differentiation, sterilization, observation, micrometers (µm), nanometers (nm), Robert Hooke, compound microscope, Antony van Leeuwenhoek, simple microscope, Royal Society of London, Father of Microbiology, Three Domain Classification, ribosomal RNA (rRNA), Bacteria, Archaea, Eukarya, obligate intracellular parasites, taxonomic ranks, genus, species, binomial nomenclature Study Questions 1. Who are the members of the microbial world? 2. What is the complete definition of microbiology? What characteristics are relevant? 3. What size are different groups of microbes? 4. What were the contributions of Hooke and Van Leeuwenhoek to the field of microbiology? How did they make these contributions? 5. What is the basis for Woese’s classification and what are the three domains? 6. What are the basic characteristics of members of the three domains? Where do microbes fit in? 7. What are the basic characteristics of viruses? Why are they not classified in one of the three domains? 8. What are taxonomic ranks? What is the system of binomial nomenclature? What are the basic rules? How are bacteria named? What is a genus and species? Be able to write a bacterial name correctly. 6 | Introduction to Microbiology 2. Microscopes You have probably figured out that microbes (AKA microorganisms) are pretty small, right? Yeah, well, size isn’t everything. But numbers, that is something. If you were to take one gram of soil and start counting the microbes in it at a rate of 1 microbe/second, it would take you over 33 years to complete your counting. Then most of you would be in your 50s and having a mid-life crisis, so let’s not go there…but the small size of microbes certainly has made it difficult to study them, particularly in the beginning. (If you want a visual idea of scale, check out the Cell Size and Scale tool, which allows you to zoom in from a coffee bean to a carbon atom. Be sure to pay careful attention to the microbes in between!) TM O.k., if you want to see something really, really, really small, who ya gonna call? Not Ghostbusters , that’s for sure. I would try someone with a microscope. (Microscope Man? Maybe not.) Now I will admit, with the advent of molecular biology there’s a lot of microbiology nowadays that happens without a microscope. But if you want to actually visualize microbes, you’ll need the ability to magnify – you’ll need a microscope of some type. And, since “seeing is believing,” it was the visualization of microbes that got people interested in them in the first place. Microscopy in the 1600s It is believed that Robert Hooke was one of the first scientists to actually observe microbes, in 1665. His illustrations and observations from a variety of objects viewed under a microscope were published in the book Micrographia. Hooke used a compound microscope, meaning it contained two sets of lenses for magnification: the ocular lens next to the eye and the objective lens, next to the specimen or object. The magnification of a compound microscope is a product of the ocular lens magnification and the objective lens magnification. Thus a microscope with an ocular magnification of 10x and an objective magnification of 50x would have a total magnification of 500x. You can see a drawing of Hooke’s microscope. Antonie van Leeuwenhoek, often called the “Father of Microbiology,” wasn’t a scientist by profession. He was a cloth merchant from Holland who was believed to be inspired by Mr. Hooke’s work, probably with the original intention of examining textiles to determine quality. Very quickly van Leeuwenhoek started examining just about everything under the microscope and we know this because he kept detailed notes about both his samples and his observations. Van Leeuwenhoek was using what is called a simple microscope, a microscope with just a single lens. Essentially, it is a magnifying glass. But the lenses that he produced were of such high quality that he is given credit for the discovery of single-celled life forms. You can learn more about van Leeuwenhoek’s observations. Microscopes | 7 Modern Microscopy: Light Microscopes Let us face it, a modern microscope is a pretty technical tool, even one of the cheaper versions. If you want to understand the limitations of a light microscope you have to understand concepts like resolution, wavelength, and numerical aperture, where their relationship to one another is summed up by the Abbé equation: In microscopy the definition of resolution is typically the ability of a lens to distinguish two objects that are close together. So, in the Abbé equation d becomes the minimal distance where two objects next to one another can be resolved or distinguished as individual objects. Resolution is dependent upon the wavelength of illumination being used, where a shorter wavelength will result in a smaller d. Lastly, we have the effect of the numerical aperture, which is a function of the objective lens and its ability to gather light. The numerical aperture value is actually defined by two components: n, which is the refractive index of the medium the lens is working in, and sin θ, which is a measurement of the cone of light that enters the objective. A lens can typically work in two media: air, with a refractive index of 1.00, or oil, with a refractive index of 1.25. Oil will allow more of the light to be collected, by directing more of the light rays into the objective lens. The maximum total magnification for a microscope using visual light for illumination is around 1500X, where the microscope might have 15x oculars and a 100x oil immersion objective. The highest resolution possible is around 0.2 μm. If objects or cells are closer together than this, they can’t be distinguished as separate entities. Here’s a nice description from Nikon, including an interactive tutorial on numerical aperture and image resolution. And then there are so many microscopes, so little time! The type that you need depends upon the specific type of microbes that you want to visualize. For light microscopes there are six different types of microscopes, all using light as the source of illumination: bright-field microscope, dark-field microscope, phase contrast microscope, differential interference contrast (DIC), fluorescence microscope, and confocal scanning laser microscope (CSLM). Let us look at the details of each type: 8 | Microscopes Bright-field Microscope The bright-field microscope is your standard microscope that you could purchase for your niece or nephew at any toy store. Here is a website on the basic parts of a bright-field compound microscope, in case you are not enrolled in the general microbiology lab. The specimen is illuminated by a light source at the base of the microscope and then initially magnified by the objective lens, before being magnified again by the ocular lens. Remember that the total magnification achieved is a product of the magnification of both lenses. The specimen is typically visualized because of differences in contrast between the specimen itself and its surrounding environment. But that does not apply to unstained bacteria, which have very little contrast with their environment, unless the cells are naturally pigmented. That is why staining (see section below) is such an important concept in microscopy. A bright-field microscope will work reasonably well to view the larger eukaryotic microbes (i.e. protozoa, algae) without stain, but unstained bacteria will be almost invisible. Stained bacteria will appear dark against a bright background (ah, I knew that there was a reason for the term “bright-field.”) Microscopes | 9 Bright-Field Microscope Dark-field Microscope The dark-field microscope is really just a slightly modified bright-field microscope. In fact, you could make this modification to the microscope you have at home! It makes use of what is known as a dark-field stop, an opaque disk that blocks light directly underneath the specimen so that light reaches it from the sides. The result is that only light that has been reflected or refracted by the specimen will be collected by the objective lens, resulting in cells that appear bright against a dark background (thus the term “dark-field.” Yes, 10 | Microscopes it’s all making sense now). This allows for observation of living, unstained cells which is particularly nice if you want to observe motility or eukaryotic organelles. Phase Contrast Microscope The phase-contrast microscope is also a modified bright-field microscope, although the modifications are getting more complex, as well as more expensive. This microscope also uses an opaque ring or annular stop, but this one has a transparent ring that only releases light in a hollow cone. The principle of this microscope gets back to the idea of refractive index and the fact that cells have a different refractive index than their surroundings, resulting in light that differs slightly in phase. The difference is amplified by a phase ring found in a special phase objective. The phase differences can be translated into differences in brightness, resulting in a dark image amidst a bright background. This allows for the observation of living, unstained cells, once again useful to observe motility or eukaryotic organelles. Phase Contrast Microscope Mechanics. Differential Interference Contrast (DIC) Microscope The differential interference contrast microscope operates on much the same principle as the phase- contrast microscope, by taking advantage of the differences in refractive index of a specimen and its surroundings. But it uses polarized light that is then split into two beams by a prism. One beam of light passes through the specimen, the other passes through the surrounding area. When the beams are Microscopes | 11 combined via a second prism they “interfere” with one another, due to being out of phase. The resulting images have an almost 3D effect, useful for observing living, unstained cells. Differential Contrast Microscope Mechanism. 12 | Microscopes Fluorescence Microscope A fluorescence microscope utilizes light that has been emitted from a specimen, rather than passing through it. A mercury-arc lamp is used to generate an intense beam of light that is filtered to produce a specific wavelength of light directed at the specimen by use of dichromatic mirror, which reflects short wavelengths and transmits longer wavelengths. Naturally fluorescent organisms will absorb the short- wavelengths and emit fluorescent light with a longer wavelength that will pass through the dichromatic mirror and can be visualized. There are a variety of microbes with natural fluorescence but there are certainly far more organisms that lack this quality. Visualization of the latter organisms depends upon the use of fluorochromes, fluorescent dyes that bind to specific cell components. The fluorochromes can also be attached to antibodies, to highlight specific structures or areas of the cell, or even different organisms. Fluorescence Microscope. By Masur (Own work) [GFDL, CC-BY-SA-3.0 or CC BY-SA 2.5-2.0-1.0], via Wikimedia Commons Microscopes | 13 Fluorescence Microscope Mechanism. Confocal Scanning Laser Microscope (CSLM) In order to understand how a confocal scanning laser microscope works, it is helpful to understand how a fluorescence microscope works, so hopefully you already read the previous section. A CSLM uses a laser for illumination, due to the high intensity. The light is directed at dichromatic mirrors that move, “scanning” the specimen. The longer wavelengths emitted by the fluorescently stained specimen pass back through 14 | Microscopes the mirrors, through a pinhole, and are measured by a detector. The pinhole serves to conjugate the focal point of the lens (ah, that’s where the term confocal came from!), which means it allows for complete focus of a given point. Since the entire specimen is scanned in the x-z planes (all three axes), the information acquired by the detector can be compiled by a computer to create a single 3D image entirely in focus. This is a particularly useful tool for viewing complex structures such as biofilms. Confocal Scanning Laser Microscope Mechanism. Staining Most of the microbes, particularly unicellular microbes, would not be apparent without the help of staining. It helps to make something so small a bit easier to see, by providing contrast between the microorganism and its background. A simple stain makes use of a single dye, either to stain the cells directly (direct stain) or to stain the background surrounding the cells (negative stain). From this a researcher can gather basic information about a cell’s size, morphology (shape), and cell arrangement. There are also more complex stains, known as differential stains, that combine stains to allow for differentiation of organisms based on their characteristics. The Gram stain, developed in 1884, is the most common differential stain used in microbiology, where bacterial cells are separated based on their cell wall type: gram positive bacteria which stain purple and gram negative bacteria which stain pink. Some bacteria have a specialized cell wall that must be stained with the acid-fast stain, where acid-fast bacteria stain red and non-acid-fast bacteria stain blue. Other differential stains target specific bacterial structures, such as endospores, capsules, and flagella, to be talked about later. Microscopes | 15 Even More Modern Microscopy: Electron Microscopes Light microscopes are great if you are observing eukaryotic microbes and they might work for observing bacteria and archaea, but they are not going to work at all to observe viruses. Remember that the limit of resolution for a light microscope is 0.2 μm or 200 nm and most viruses are smaller than that. So, we need something more powerful. Enter the electron microscopes, which replace light with electrons for visualization. Since electrons have a wavelength of 1.23 nm (as opposed to the 530 nm wavelength of blue- green light), resolution increases to around 0.5 nm, with magnifications over 150,000x. The drawback of using electrons is that they must be contained in a vacuum, eliminating the possibility of working with live cells. There is also some concern that the extensive sample preparation might distort the specimen’s characteristics or cause artifacts to form. There are two different types of electron microscope, the transmission electron microscope (TEM) and the scanning electron microscope (SEM): Transmission Electron Microscope (TEM) The transmission electron microscope utilizes an electron beam directed at the specimen with the use of electromagnets. Dense areas scatter the electrons, resulting in a dark area on the image, while electrons can pass (or “transmit”) through the less dense areas, resulting in a brighter section. The image is generated on a fluorescent screen and can then be captured. Since the electrons are easily scattered by extremely thick specimens, samples must be sliced down to 20-100 nm in thickness, typically by being embedded in some type of plastic and then being cut with a diamond knife into extremely thin sections. The resulting pictures represent one slice or plane of the specimen. 16 | Microscopes Transmission Electron Microscope. Microscopes | 17 18 | Microscopes Transmission Electron Microscope. By kallerna (Own work) [Public domain], via Wikimedia Commons Scanning Electron Microscope (SEM) The scanning electron microscope also utilizes an electron beam but the image is formed from secondary electrons that were released from the surface of the specimen and then collected by a detector. More electrons are released from raised areas of the specimen, while less secondary electrons will be collected from sunken areas. In addition, the electron beam is scanned over the specimen surface, producing a 3D image of the external features. If you want to see some beautiful TEM and SEM photomicrographs, check out Dennis Kunkel’s site. Most have been colorized, but they are quite stunning. On the other end of the spectrum, here are pictures taken with the Intel Play QX3, a plastic microscope for children. Be careful, you could get lost in this website. But it is great to see what an inexpensive microscope can produce in the hands of someone who knows what they’re doing! These pictures are stunning as well. Scanning Electron Microscope Mechanism. Microscopes | 19 Scanning Electron Microscope. By en:User:Olaboy [CC BY-SA 2.5], Via Wikipedia Commons Welcome to 21st Century Microscopy: Scanning Probe Microscopes As technology has advanced, even more powerful microscopes have been invented, ones that can even allow for visualization at the atomic level. These microscopes can be used in microbiology but more often they are used in other fields, to allow visualization of chemicals, metals, magnetic samples, and nanoparticles, wherever the 0.1 nm resolution and 100,000,000x magnification might be needed. The scanning probe microscopes are thus named because they move some type of probe over a specimen’s surface in the x-z planes, allowing computers to generate an extremely detailed 3D image of the specimen. Resolution is so high because the probe size is much smaller than the wavelength of either visible light or electrons. Both microscopes can be used to study objects in liquid, allowing for the examination of biological molecules. There are two different types of scanning probe microscopes, the scanning tunneling microscope (STM) and the atomic force microscope (AFM): 20 | Microscopes Scanning Tunneling Microscope (STM) The scanning tunneling microscope has an extremely sharp probe, 1 atom thick, that maintains a constant voltage with the specimen surface allowing electrons to travel between them. This tunneling current is maintained by raising and lowering the probe to sustain a constant height above the sample. The resulting motion is tracked by a computer, which generates the final image. Scanning Tunneling Microscope Mechanism. Atomic Force Microscope (AFM) The atomic force microscope was developed as an alternative to the STM, for use with samples that do not conduct electricity well. The microscope utilizes a cantilever with an extremely sharp probe tip that maintains a constant height above the specimen, typically by direct contact with the sample. Movement of the cantilever to maintain this contact deflects a laser beam, translating into an image of the object. Once again, computers are used to generate the image. Microscopes | 21 Atomic Force Microscope Mechanism. Key Words magnification, Robert Hooke, compound microscope, ocular lens, objective lens, total magnification, van Leeuwenhoek, simple microscope, Abbé equation, resolution, wavelength, numerical aperture, refractive index, oil immersion objective, light microscope, bright-field microscope, dark-field microscope, phase contrast microscope, differential interference contrast (DIC) microscope, fluorescence microscope, fluorochrome, confocal scanning laser microscope (CSLM), simple stain, direct stain, negative stain, differential stain, Gram stain, acid-fast stain, electron microscope (EM), transmission electron microscope (TEM), scanning electron microscope (SEM), scanning probe microscopes, scanning tunneling microscopes (STM), atomic force microscope (AFM). 22 | Microscopes Study Questions 1. What roles did Hooke and van Leeuwenhoek play in the development of microscopy? How did their contributions differ? 2. How do magnification & resolution differ? How is total magnification determined? 3. How does the Abbé equation explain the resolution of a microscope? What components impact resolution and in what way? What is the function of immersion oil? 4. Know the main uses and understand the principle mechanics of the following light microscopes: brightfield, dark-field, phase contrast, fluorescence, and differential interference contrast. 5. How does the confocal scanning laser microscope work to form a 3 dimensional image? How does it improve resolution compared to other light microscopes? 6. How is staining used in microscopy? What are the general categories of stains and how are they used? 7. What are the advantages and problems with electron microscopes? What are the effective magnification, resolution and main uses of electron microscopes? 8. How does the TEM differ from the SEM in terms of function and final product? 9. How do scanning probe microscopes work and what do they allow us to see? Why are they useful for studying biological molecules? What is the difference in the scanning tunneling and the atomic force microscope? Exploratory Questions (OPTIONAL) 1. How have microscopes improved our understanding of microbes? What are the limitations of microscopes and the information that we get from them? Microscopes | 23 3. Cell Structure You say “procaryote,” I say “prokaryote,” you say “eucaryote,” I say “eukaryote”….well, however, you spell them, it’s the next topic. Traditionally, cellular organisms have been divided into two broad categories, based on their cell type. They are either prokaryotic or eukaryotic. In general, prokaryotes are smaller, simpler, with a lot less stuff, which would make eukaryotes larger, more complex, and more cluttered. The crux of their key difference can be deduced from their names: “karyose” is a Greek word meaning “nut” or “center,” a reference to the nucleus of a cell. “Pro” means “before,” while “Eu” means “true,” indicating that prokaryotes lack a nucleus (“before a nucleus”) while eukaryotes have a true nucleus. More recently, microbiologists have been rebelling against the term prokaryote because it lumps both bacteria and the more recently discovered archaea in the same category. Both cells are prokaryotic because they lack a nucleus and other organelles (such as mitochondria, Golgi apparatus, endoplasmic reticulum, etc), but they aren’t closely related genetically. So, to honor these differences this text will refer to the groups as the archaea, the bacteria, and the eukaryotes and try to leave the prokaryotic reference out of it. Cell Morphology Cell morphology is a reference to the shape of a cell. It might seem like a trivial concept but to a cell it is not. The shape dictates how that cell will grow, reproduce, obtain nutrients, move, and it’s important to the cell to maintain that shape to function properly. Cell morphology can be used as a characteristic to assist in identifying particular microbes but it’s important to note that cells with the same morphology are not necessarily related. Bacteria tend to display the most representative cell morphologies, with the most common examples listed here: Bacterial Cell Morphologies. 24 | Cell Structure Coccus (pl. cocci) – a coccus is a spherically shaped cell. Bacillus (pl. bacilli) – a bacillus is a rod-shaped cell. Curved rods – obviously this is a rod with some type of curvature. There are three sub-categories: the vibrio, which are rods with a single curve and the spirilla/spirochetes, which are rods that form spiral shapes. Spirilla and spirochetes are differentiated by the type of motility that they exhibit, which means it is hard to separate them unless you are looking at a wet mount. Pleomorphic – pleomorphic organisms exhibit variability in their shape. There are additional shapes seen for bacteria, and an even wider array for the archaea, which have even been found as star or square shapes. Eukaryotic microbes also tend to exhibit a wide array of shapes, particularly the ones that lack a cell wall such as the protozoa. Cell Size Cell size, just like cell morphology, is not a trivial matter either, to a cell. There are reasons why most archaeal/bacterial cells are much smaller than eukaryotic cells. Much of it has to do with the advantages derived from being small. These advantages relate back to the surface-to-volume ratio of the cell, a ratio of the external cellular layer in contact with the environment compared to the liquid inside. This ratio changes as a cell increases in size. Let us look at a 2 μm cell in comparison with a cell that is twice as large at 4 μm. r = 2 μm surface area = 50.3 μm2 r = 1 μm volume = 33.5 μm3 surface area = 12.6 μm2 volume = 4.2 μm3 The surface-to-volume ratio of the smaller cell is 3, while the surface-to-volume ratio of the larger cell decreases to 1.5. Think of the cell surface as the ability of the cell to bring in nutrients and let out waste products. The larger the surface area, the more possibilities exist for engaging in these activities. Based on this, the larger cell would have an advantage. Now think of the volume as representing what the cell has to support. As the surface-to-volume ratio decreases, the larger cell will struggle to bring in the nutrients necessary to support the cell’s activities at a rapid rate, such as growth and reproduction, despite the larger surface area. Thus, small cells grow and reproduce faster than larger cells, despite having a smaller surface area. So, small cells grow and reproduce faster. This also means that they evolve faster over time, giving them more opportunities to adapt to environments. Keep in mind that the size difference (bacterial/archaeal cells = smaller, eukaryotic cells = larger) is on average. A typical bacterial/archaeal cell is a few micrometers in size, while a typical eukarytotic cell is about 10x larger. There are a few monster bacteria that fall outside the norm in size and still manage to grow and reproduce very quickly. One such example is Thiomargarita namibiensis, which can measure from 100-750 μm in length, compared to the more typical 4 μm length of E. coli. T. namibiensis manages to maintain its rapid reproductive rate by producing very large vacuoles or bubbles that occupy a large portion Cell Structure | 25 of the cell. These vacuoles reduce the volume of the cell, increasing the surface-to-volume ratio. Other very large bacteria utilize a ruffled membrane for their outer surface layer. This increases the surface area, which also increase the surface-to-volume ratio, allowing the cell to maintain its rapid reproductive rate. Cell Components All cells (bacterial, archaeal, eukaryotic) share four common components: Cytoplasm – cytoplasm is the gel-like fluid that fills each cell, providing an aqueous environment for the chemical reactions that take place in a cell. It is composed of mostly water, with some salts and proteins. DNA – deoxyribonucleic acid or DNA is the genetic material of the cell, the instructions for the cell’s abilities and characteristics. This complete set of genes, referred to as a genome, is localized in an irregularly-shaped region known as the nucleoid in bacterial and archaeal cells, and enclosed into a membrane-bound nucleus in eukaryotic cells. Ribosomes – the protein-making factories of the cell are the ribosomes. Composed of both RNA and protein, there are some distinct differences between the ones found in bacteria/archaea and the ones found in eukaryotes, particularly in terms of size and location. The ribosomes of bacteria and archaea are found floating in the cytoplasm, while many of the eukaryotic ribosomes are organized along the endoplasmic reticulum, a eukaryotic organelle. Ribosomes are measured using the Svedberg unit, which corresponds to the rate of sedimentation when centrifuged. Bacterial/archaeal ribosomes have a measurement of 70S as a sedimentation value, while eukaryotic ribosomes have a measurement of 80S, an indication of both their larger size and mass. Cell Membrane – one of the outer boundaries of every cell is the cell membrane. A plasma membrane can be found elsewhere as well, such as the membrane that bounds the eukaryotic nucleus, while the term cell membrane refers specifically to this boundary of the cell proper. The plasma membrane separates the cell’s inner contents from the surrounding environment. While not a strong layer, the plasma membrane participates in several crucial processes for the cell, particularly for bacteria and archaea, which typically only have the one membrane: ◦ Acts as a semi-permeable barrier to allow for the entrance and exit of select molecules. It functions to let in nutrients, excrete waste products, and possibly keep out dangerous substances such as toxins or antibiotics. ◦ Performs metabolic processes by participating in the conversion of light or chemical energy into a readily useable form known as ATP. This energy conservation involves the development of a proton motive force (PMF), based on the separation of charges across the membrane, much like a battery. ◦ “Communicates” with the environment by binding or taking in small molecules that act as signals and provide information important to the cell. The information might relate to nutrients or toxin in the area, as well as information about other organisms. 26 | Cell Structure Typical Prokaryotic Cell. Cell Structure | 27 Typical Eukaryotic Cell. By Mediran (Own work) [CC BY-SA 3.0], via Wikimedia Commons Eukaryotes have numerous additional components called organelles, such as the nucleus, the mitochrondria, the endoplasmic reticulum, the Golgi apparatus, etc. These are all membrane-bound compartments that house different activities for the cell. Because each structure is bounded by its very own plasma membrane, it provides the cell with multiple locations for membranous functions to occur. Plasma Membrane Structure When talking about the details of the plasma membrane it gets a little bit complicated, since bacteria and eukaryotes share the same basic structure, while archaea have marked differences. For now let us cover the basic structure, while the archaeal modifications and variations will be covered in the chapter on archaea. The plasma membrane is often described by the fluid-mosaic model, which accounts for the movement of various components within the membrane itself. The general structure is explained by the separation of individual substances based on their attraction or repulsion of water. The membrane is typically composed of two layers (a bilayer) of phospholipids, which form the basic structure. Each phospholipid is composed of a polar region that is hydrophilic (“water loving”) and a non-polar region that is hydrophobic (“water fearing”). The phospholipids will spontaneously assemble in such a way as to keep the polar regions in contact with the aqueous environment outside of the cell and the cytoplasm inside, while the non-polar regions are sequestered in the middle, much like the jelly in a sandwich. 28 | Cell Structure The phospholipids themselves are composed of a negatively-charged polar head which is a phosphate group, connected by a glycerol linkage to two fatty acid tails. The phosphate group is hydrophilic while the fatty acid tails are hydrophobic. While the membrane is not considered to be particularly strong, it is strengthened somewhat by the presence of additional lipid components, such as the steroids in eukaryotes and the sterol-like hopanoids in bacteria. Embedded and associated with the phospholipid bilayer are various proteins, with myriad functions. Proteins that are embedded within the bilayer itself are called integral proteins while proteins that associate on the outside of the membrane are called peripheral proteins. Some of the peripheral proteins are anchored to the membrane via a lipid tail, and many associate with specific integral proteins to fulfill cellular functions. Integral proteins are the dominant type, representing about 70-80% of the proteins associated with a plasma membrane, while the peripheral proteins represent the remaining 20-30%. Plasma Membrane Structure. The amount of protein composing a plasma membrane, in comparison to phospholipid, differs by organism. Bacteria have a very high protein to phospholipid ratio, around 2.5:1, while eukaryotes exhibit a ratio of 1:1, at least in their cell membrane. But remember that eukaryotes have multiple plasma membranes, one for every organelle. The protein to phospholipid ratio for their mitochondrial membrane is 2.5:1, just like the bacterial plasma membrane, providing additional evidence for the idea that eukaryotes evolved from a bacterial ancestor. Key Words prokaryote, eukaryote, morphology, coccus, bacillus, vibrio, spirilla, spirochete, pleomorphic, surface-to- volume (S/V) ratio, cytoplasm, DNA, genome, nucleoid, nucleus, ribosome, Svedberg unit, plasma membrane, cell membrane, proton motive force (PMF), fluid-mosaic model, phospholipid, hydrophilic, hydrophobic, polar head, phosphate group, glycerol linkage, fatty acid tail, steroids, hopanoids, phospholipid bilayer, integral protein, peripheral protein. Cell Structure | 29 Study Questions 1. Why are microbiologists questioning the traditional ways of thinking about “prokaryotes”? 2. What are the 3 basic shapes of Bacteria? 3. How do microbes belonging to the categories of Eukaryotes vs. Bacteria/Archaea typically differ in terms of size? How does size impact a cell? How does surface-to-volume ratio influence cell size and capabilities? How can cells get around limitations imposed by a smaller surface:volume ratio? 4. What are two ways that bacteria can adapt to being large? Give specific examples. 5. What are the basic components of any cell? 6. What are the roles of the plasma membrane? 7. What is the fluid mosaic model? 8. Understand the basic structure of the phospholipids of the plasma membrane and the role that it plays in membrane design. 9. What are other lipids found in the plasma membrane? 10. What are the 2 categories of proteins found in the plasma membrane and how do they differ? 11. How do the phospholipids and the protein come together to form a working plasma membrane? 12. What is the importance of the protein to phospholipid ratio in terms of evolution? Exploratory Questions (OPTIONAL) 1. What is the largest bacterium or archaean ever discovered? What is the smallest eukaryote ever discovered? 30 | Cell Structure 4. Bacteria: Cell Walls It is important to note that not all bacteria have a cell wall. Having said that though, it is also important to note that most bacteria (about 90%) have a cell wall and they typically have one of two types: a gram positive cell wall or a gram negative cell wall. The two different cell wall types can be identified in the lab by a differential stain known as the Gram stain. Developed in 1884, it’s been in use ever since. Originally, it was not known why the Gram stain allowed for such reliable separation of bacterial into two groups. Once the electron microscope was invented in the 1940s, it was found that the staining difference correlated with differences in the cell walls. Here is a website that shows the actual steps of the Gram stain. After this stain technique is applied the gram positive bacteria will stain purple, while the gram negative bacteria will stain pink. Overview of Bacterial Cell Walls A cell wall, not just of bacteria but for all organisms, is found outside of the cell membrane. It’s an additional layer that typically provides some strength that the cell membrane lacks, by having a semi-rigid structure. Both gram positive and gram negative cell walls contain an ingredient known as peptidoglycan (also known as murein). This particular substance hasn’t been found anywhere else on Earth, other than the cell walls of bacteria. But both bacterial cell wall types contain additional ingredients as well, making the bacterial cell wall a complex structure overall, particularly when compared with the cell walls of eukaryotic microbes. The cell walls of eukaryotic microbes are typically composed of a single ingredient, like the cellulose found in algal cell walls or the chitin in fungal cell walls. The bacterial cell wall performs several functions as well, in addition to providing overall strength to the cell. It also helps maintain the cell shape, which is important for how the cell will grow, reproduce, obtain nutrients, and move. It protects the cell from osmotic lysis, as the cell moves from one environment to another or transports in nutrients from its surroundings. Since water can freely move across both the cell membrane and the cell wall, the cell is at risk for an osmotic imbalance, which could put pressure on the relatively weak plasma membrane. Studies have actually shown that the internal pressure of a cell is similar to the pressure found inside a fully inflated car tire. That is a lot of pressure for the plasma membrane to withstand! The cell wall can keep out certain molecules, such as toxins, particularly for gram negative bacteria. And lastly, the bacterial cell wall can contribute to the pathogenicity or disease –causing ability of the cell for certain bacterial pathogens. Bacteria: Cell Walls | 31 Structure of Peptidoglycan Let us start with peptidoglycan, since it is an ingredient that both bacterial cell walls have in common. Peptidoglycan is a polysaccharide made of two glucose derivatives, N-acetylglucosamine (NAG) and N- acetylmuramic acid (NAM), alternated in long chains. The chains are cross-linked to one another by a tetrapeptide that extends off the NAM sugar unit, allowing a lattice-like structure to form. The four amino acids that compose the tetrapeptide are: L-alanine, D-glutamine, L-lysine or meso-diaminopimelic acid (DPA), and D-alanine. Typically only the L-isomeric form of amino acids are utilized by cells but the use of the mirror image D-amino acids provides protection from proteases that might compromise the integrity of the cell wall by attacking the peptidoglycan. The tetrapeptides can be directly cross-linked to one another, with the D-alanine on one tetrapeptide binding to the L-lysine/ DPA on another tetrapeptide. In many gram positive bacteria there is a cross-bridge of five amino acids such as glycine (peptide interbridge) that serves to connect one tetrapeptide to another. In either case the cross-linking serves to increase the strength of the overall structure, with more strength derived from complete cross-linking, where every tetrapeptide is bound in some way to a tetrapeptide on another NAG-NAM chain. While much is still unknown about peptidoglycan, research in the past ten years suggests that peptidoglycan is synthesized as a cylinder with a coiled substructure, where each coil is cross-linked to the coil next to it, creating an even stronger structure overall. Peptidoglycan Structure. Gram Positive Cell walls The cell walls of gram positive bacteria are composed predominantly of peptidoglycan. In fact, peptidoglycan can represent up to 90% of the cell wall, with layer after layer forming around the cell membrane. The NAM tetrapeptides are typically cross-linked with a peptide interbridge and complete cross-linking is common. All of this combines together to create an incredibly strong cell wall. 32 | Bacteria: Cell Walls The additional component in a gram positive cell wall is teichoic acid, a glycopolymer, which is embedded within the peptidoglycan layers. Teichoic acid is believed to play several important roles for the cell, such as generation of the net negative charge of the cell, which is essential for development of a proton motive force. Teichoic acid contributes to the overall rigidity of the cell wall, which is important for the maintenance of the cell shape, particularly in rod-shaped organisms. There is also evidence that teichoic acids participate in cell division, by interacting with the peptidoglycan biosynthesis machinery. Lastly, teichoic acids appear to play a role in resistance to adverse conditions such as high temperatures and high salt concentrations, as well as to β-lactam antibiotics. Teichoic acids can either be covalently linked to peptidoglycan (wall teichoic acids or WTA) or connected to the cell membrane via a lipid anchor, in which case it is referred to as lipoteichoic acid. Since peptidoglycan is relatively porous, most substances can pass through the gram positive cell wall with little difficulty. But some nutrients are too large, requiring the cell to rely on the use of exoenzymes. These extracellular enzymes are made within the cell’s cytoplasm and then secreted past the cell membrane, through the cell wall, where they function outside of the cell to break down large macromolecules into smaller components. Gram Negative Cell Walls The cell walls of gram negative bacteria are more complex than that of gram positive bacteria, with more ingredients overall. They do contain peptidoglycan as well, although only a couple of layers, representing 5-10% of the total cell wall. What is most notable about the gram negative cell wall is the presence of a plasma membrane located outside of the peptidoglycan layers, known as the outer membrane. This makes Bacteria: Cell Walls | 33 up the bulk of the gram negative cell wall. The outer membrane is composed of a lipid bilayer, very similar in composition to the cell membrane with polar heads, fatty acid tails, and integral proteins. It differs from the cell membrane by the presence of large molecules known as lipopolysaccharide (LPS), which are anchored into the outer membrane and project from the cell into the environment. LPS is made up of three different components: 1) the O-antigen or O-polysaccharide, which represents the outermost part of the structure , 2) the core polysaccharide, and 3) lipid A, which anchors the LPS into the outer membrane. LPS is known to serve many different functions for the cell, such as contributing to the net negative charge for the cell, helping to stabilize the outer membrane, and providing protection from certain chemical substances by physically blocking access to other parts of the cell wall. It is also known that LPS can play a role in the response that a host (think human or other animal) develops to an infection by a pathogenic gram negative bacterium. Specifically, the O-antigen portion of LPS triggers an immune response in the host, causing the generation of protective antibodies that identify a particular O-antigen (think of E. coli O157: H7). It is also known that lipid A can act as a toxin in humans, specifically an endotoxin, causing general symptoms of illness such as fever and diarrhea. A large amount of lipid A released into the bloodstream can trigger endotoxic shock, a body-wide inflammatory response which can be life-threatening. The outer membrane does present an obstacle for the cell. While there are certain molecules it would like to keep out, such as antibiotics and toxic chemicals, there are nutrients that it would like to let in and the additional lipid bilayer presents a formidable barrier. Large molecules are broken down by enzymes, in order to allow them to get past the LPS. Instead of exoenzymes (like the gram positive bacteria), the gram negative bacteria utilize periplasmic enzymes that are stored in the periplasm. Where is the periplasm, you ask? It is the space located between the outer surface of the cell membrane and the inner surface of the outer membrane, and it contains the gram negative peptidoglycan. Once the periplasmic enzymes have broken nutrients down to smaller molecules that can get past the LPS, they still need to be transported across the outer membrane, specifically the lipid bilayer. Gram negative cells utilize porins, which are 34 | Bacteria: Cell Walls transmembrane proteins composed of a trimer of three subunits, which form a pore across the membrane. Some porins are non-specific and transport any molecule that fits, while some porins are specific and only transport substances that they recognize by use of a binding site. Once across the outer membrane and in the periplasm, molecules work their way through the porous peptidoglycan layers before being transported by integral proteins across the cell membrane. The peptidoglycan layers are linked to the outer membrane by the use of a lipoprotein known as Braun’s lipoprotein (good ol’ Dr. Braun). At one end the lipoprotein is covalently bound to the peptidoglycan while the other end is embedded into the outer membrane via its polar head. This linkage between the two layers provides additional structural integrity and strength. Unusual and Wall-less Bacteria Having emphasized the important of a cell wall and the ingredient peptidoglycan to both the gram positive and the gram negative bacteria, it does seem important to point out a few exceptions as well. Bacteria belonging to the phylum Chlamydiae appear to lack peptidoglycan, although their cell walls have a gram negative structure in all other regards (i.e. outer membrane, LPS, porin, etc). It has been suggested that they might be using a protein layer that functions in much the same way as peptidoglycan. This has an advantage to the cell in providing resistance to β-lactam antibiotics (such as penicillin), which attack peptidoglycan. Bacteria belonging to the phylum Tenericutes lack a cell wall altogether, which makes them extremely susceptible to osmotic changes. They often strengthen their cell membrane somewhat by the addition of sterols, a substance usually associated with eukaryotic cell membranes. Many members of this phylum are pathogens, choosing to hide out within the protective environment of a host. Key Words cell wall, gram positive bacteria, gram negative bacteria, Gram stain, peptidoglycan, murein, osmotic lysis, N- acetylglucosamine (NAG), N-acetylmuramic acid (NAM), tetrapeptide, L-alanine, D-glutamine, L-lysine, meso-diaminopimelic acid (DPA), D-alanine, direct cross-link, peptide interbridge, complete cross-linking, teichoic acid, wall teichoic acid (WTA), lipoteichoic acid, exoenzymes, outer membrane, lipopolysaccharide (LPS), O-antigen or O-polysaccharide, core polysaccharide, lipid A, endotoxin, periplasmic enzymes, periplasm, porins, Braun’s lipoprotein, Chlamydiae, Tenericutes, sterols. Study Questions 1. What are the basic characteristics and functions of the cell wall in Bacteria? 2. What is the Gram stain and how does it relate to the different cell wall types of Bacteria? Bacteria: Cell Walls | 35 3. What is the basic unit structure of peptidoglycan? What components are present and how do they interact? Be able to diagram peptidoglycan and its’ components. 4. What is cross linking and why does this play such an important role in the cell wall? What different types of cross-linking are there? 5. Why are D-amino acids unusual and how does having D-amino acids in the peptidoglycan keep this macromolecule stable? 6. What are the differences between gram positive and negative organisms in terms of thickness of peptidoglycan, different constituents of PG and variations in cross linkage and strength, and other molecules associated with cell wall? 7. What is teichoic acid and what are its’ proposed roles and functions? What are lipteichoic acids? 8. What is the periplasm of gram negative bacteria? What purpose can it serve? What alternatives are available for cells? 9. What is the general composition of the outer membrane of gram-negative microorganisms, its function and toxic properties? How is it linked to the cell? What is a porin and what are their functions? 10. What group of bacteria lack peptidoglycan in their cell wall? What advantage does this confer? 11. What group of bacteria normally does not have cell walls and how do they maintain themselves? Exploratory Questions (OPTIONAL) 1. How does the mechanism of the Gram stain relate to specific components of the bacterial cell wall? 36 | Bacteria: Cell Walls 5. Bacteria: Internal Components We have already covered the main internal components found in all bacteria, namely, cytoplasm, the nucleoid, and ribosomes. Remember that bacteria are generally thought to lack organelles, those bilipid membrane-bound compartments so prevalent in eukaryotic cells (although some scientists argue that bacteria possess structures that could be thought of as simple organelles). But bacteria can be more complex, with a variety of additional internal components to be found that can contribute to their capabilities. Most of these components are cytoplasmic but some of them are periplasmic, located in the space between the cytoplasmic and outer membrane in gram negative bacteria. Cytoskeleton It was originally thought that bacteria lacked a cytoskeleton, a significant component of eukaryotic cells. In the last 20 years, however, scientists have discovered bacterial filaments made of proteins that are analogues to the cytoskeletal proteins found in eukaryotes. It has also been determined that the bacterial cytoskeleton plays important roles in cell shape, cell division, and integrity of the cell wall. FtsZ FtsZ, homologous to the eukaryotic protein tubulin, forms a ring structure in the middle of the cell during cell division, attracting other proteins to the area in order to construct a septum that will eventually separate the two resulting daughter cells. MreB MreB, homologous to the eukaryotic protein actin, is found in bacillus and spiral-shaped bacteria and plays an essential role in cell shape formation. MreB assumes a helical configuration running the length of the cell and dictates the activities of the peptidoglycan-synthesis machinery, assuring a non-spherical shape. Crescentin Crescentin, homologous to the eukaryotic proteins lamin and keratin, is found in spiral-shaped bacteria with a single curve. The protein assembles lengthwise in the inner curvature of the cell, bending the cell into its final shape. Bacteria: Internal Components | 37 Cytoskeleton Structures. Inclusions Bacterial inclusions are generally defined as a distinct structure located either within the cytoplasm or periplasm of the cell. They can range in complexity, from a simple compilation of chemicals such as crystals, to fairly complex structures that start to rival that of the eukaryotic organelles, complete with a membranous external layer. Their role is often to store components as metabolic reserves for the cell when a substance is found in excess, but they can also play a role in motility and metabolic functions as well. Carbon storage Carbon is the most common substance to be stored by a cell, since all cells are carbon based. In addition, carbon compounds can often be broken down quickly by the cell, so they can serve as energy sources as well. One of the simplest and most common inclusions for carbon storage is glycogen, in which glucose units are linked together in a multi-branching polysaccharide structure. Another common way for bacteria to store carbon is in the form of poly-β-hydroxybutyrate (PHB), a granule that forms when β-hydroxybutyric acid units aggregate together. This lipid is very plastic-like in composition, leading some scientists to investigate the possibility of using them as a biodegradeable plastic. The PHB granules actually have a shell composed of both protein and a small amount of phospholipid. Both glycogen and PHB are formed when there is an excess of carbon and then broken down by the cell later for both carbon and energy. 38 | Bacteria: Internal Components Inorganic storage Often bacteria need something other than carbon, either for synthesis of cell components or as an alternate energy reserve. Polyphosphate granules allow for the accumulation of inorganic phosphate (PO43-), where the phosphate can be used to make nucleic acid (remember the sugar-phosphate backbone?) or ATP (adenosine triphosphate, of course). Other cells need sulfur as a source of electrons for their metabolism and will store excess sulfur in the form of sulfur globules, which result when the cell oxidizes hydrogen sulfide (H2S) to elemental sulfur (S0), resulting in the formation of refractile inclusions. Non-storage functions There are times when a bacterium needs to do something beyond simple storage of organic or inorganic compounds for use in metabolism and there are inclusions to help with these non-storage functions. One such example is gas vacuoles, which are used by the cell to control buoyancy in a water column, providing the cell with some control over where it is in the environment. It is a limited form of motility, on the vertical axis only. Gas vacuoles are composed of conglomerations of gas vesicles, cylindrical structures that are both hollow and rigid. The gas vesicles are freely permeable to all types of gases by passive diffusion and can be quickly constructed or collapsed, as needed by the cell to ascend or descend. Magnetosomes are inclusions that contain long chains of magnetite (Fe3O4), which are used by the cell as a compass in geomagnetic fields, for orientation within their environment. Magnetotactic bacteria are typically microaerophilic, preferring an environment with a lower level of oxygen than the atmosphere. The magenetosome allows the cells to locate the optimum depth for their growth. Magenetosomes have a true lipid bilayer, reminiscent of eukaryotic organelles, but it is actually an invagination of the cell’s plasma membrane that has been modified with specific proteins. Microcompartments Bacterial microcompartments (BMCs) are unique from other inclusions by virtue of their structure and functionality. They are icosahedral in shape and composed of a protein shell made up of various proteins in the BMC family. While their exact role varies, they all participate in functions beyond simple storage of substances. These compartments provide both a location and the substances (usually enzymes) necessary for particular metabolic activities. The best studied example of a BMC is the carboxysome, which are found in many CO2-fixing bacteria. Carboxysomes contain the enzyme ribulose-1,5-bisphosphate carboxylase (luckily it is also known as RubisCO), which plays a crucial role in converting CO2 into sugar. The carboxysome also plays a role in the concentration of CO2, thus ensuring that the components necessary for CO2-fixation are all in the same place at the same time. Bacteria: Internal Components | 39 Anammoxosome The anammoxosome is a large membrane-bound compartment found in bacterial cells capable of carrying out the anammox reaction (anaerobic ammonium oxidation), where ammonium (NH4+) and nitrite (NO2-) are converted to dinitrogen gas (N2). The process is performed as a way for the cell to get energy, using ammonium as an electron donor and nitrite as an electron accept, with the resulting production of nitrogen gas. This chemical conversion of nitrogen is important for the nitrogen cycle. Nitrogen Cycle. By Shou-Qing Ni and Jian Zhang [CC BY 3.0], via Wikimedia Commons Chlorosome Found in some phototrophic bacteria, a chlorosome is a highly efficient structure for capturing low light intensities. Lining the inside perimeter of the cell membrane, each chlorosome can contain up to 250,000 bacteriochlorophyll molecules, arranged in dense arrays. Harvested light is transferred to reaction centers in the cell membrane, allowing the conversion from light energy to chemical energy in the form of ATP. The chlorosome is bounded by a lipid monolayer. 40 | Bacteria: Internal Components Plasmid A plasmid is an extrachromosomal piece of DNA that some bacteria have, in addition to the genetic material found in the nucleoid. It is composed of double-stranded DNA and is typically circular, although linear plasmids have been found. Plasmids are described as being “non-essential” to the cell, where the cell can function normally in their absence. But while plasmids have only a few genes, they can confer important capabilities for the cell, such as antibiotic resistance. Plasmids replicate independently of the cell and can be lost (known as curing), either spontaneously or due to exposure to adverse conditions, such as UV light, thymine starvation, or growth above optimal conditions. Some plasmids, known as episomes, can be integrated into the cell chromosome where the genes will be replicated during cell division. Endospore Then there is the endospore, a marvel of bacterial engineering. This is located under the heading “bacterial internal components,” but it is important to note that an endospore isn’t an internal or external structure so much as a conversion of the cell into an alternate form. Cells start out as a vegetative cell, doing all the things a cell is supposed to do (metabolizing, reproducing, mowing the lawn…). If they get exposed to hostile conditions (dessication, high heat, an angry neighbor…) and they have the ability, they might convert from a vegetative cell into an endospore. The endospore is actually formed within the vegetative cell (doesn’t that make it an internal structure?) and then the vegetative cell lyses, releasing the endospore (does that make it an external structure?). Endospore Layers. Endospores are only formed by a few gram positive genera and provide the cell with resistance to a wide Bacteria: Internal Components | 41 variety of harsh conditions, such as starvation, extremes in temperature, exposure to drying, UV light, chemicals, enzymes, and radiation. While the vegetative cell is the active form for bacterial cells (growing, metabolizing, etc), the endospore can be thought of as a dormant form of the cell. It allows for survival of adverse conditions, but it does not allow the cell to grow or reproduce. Structure In order to be so incredibly resistant to so many different substances and environmental conditions, many different layers are necessary. The bacterial endospore has many different layers, starting with a core in the center. The core is the location of the nucleoid, ribosomes, and cytoplasm of the cell, in an extremely dehydrated form. It typically contains only 25% of the water found in a vegetative cell, increasing heat resistance. The DNA is further protected by the presence of small acid-soluble proteins (SASPs), which stabilize the DNA and protect it from degradation. DNA stabilization is increased by the presence of dipicolinic acid complexed with calcium (Ca-DPA), which inserts between the DNA bases. The core is wrapped in an inner membrane that provides a permeability barrier to chemicals, which is then surrounded by the cortex, a thick layer consisting of peptidoglycan with less cross-linking than is found in the vegetative cell. The cortex is wrapped in an outer membrane. Lastly are several spore coats made of protein, which provide protection from environmental stress such as chemicals and enzymes. Sporulation: conversion from vegetative cell to endospore Sporulation, the conversion of vegetative cell into the highly protective endospore, typically occurs when the cell’s survival is threatened in some way. The actual process is very complex and typically takes several hours until completion. Initially the sporulating cells replicates its DNA, as if it were about to undergo cell division. A septum forms asymmetrically, sequestering one copy of the chromosome at one end of the cell (called the forespore). Synthesis of endospore-specific substances occurs, altering the forespore and leading to the development of the layers specific for an endospore, as well as dehydration. Eventually the “mother cell” is lyses, allowing for the release of the mature endospore into the environment. 42 | Bacteria: Internal Components Sporulation. Conversion from endospore to vegetative cell The endospore remains dormant until environmental conditions improve, causing a chemical change that initiates gene expression. There are three distinct stages in the conversion from an endospore to the metabolically active vegetative cells: 1) activation, a preparation step that can be initiated by the application of heat; 2) germination, when the endospore becomes metabolically active and begins to take on water; 3) outgrowth, when the vegetative cell fully emerges from the endospore shell. Key Words cytoskeleton, FtsZ, tubulin, MreB, actin, crescentin, lamin, keratin, inclusion, glycogen, poly-β- hydroxybutyrate (PHB), polyphosphate granule, sulfur globule, gas vacuole, gas vesicle, magnetosome, microaerophilic, microcompartment, bacterial microcompartments (BMCs), carboxysome, ribulose-1,5-bisphosphate carboxylase, RubisCO, anammoxosome, anammox reaction, chlorosome, plasmid, curing, episome, endospore, vegetative cell, core, small acid-soluble proteins (SASPs), dipicolinic acid, Ca-DPA, inner membrane, cortex, outer membrane, spore coat, sporulation, forespore, activation, germination, outgrowth. Bacteria: Internal Components | 43 Study Questions 1. What are the roles and composition of the bacterial cytoskeleton? How does it differ from the eukaryotic cytoskeleton? What are the specific bacterial cytoskeleton proteins and what details are known about each? 2. What is the purpose of inclusions found in bacteria? What are their characteristics? 3. What are the specific examples of storage inclusions found in bacteria? Be able to describe each type in terms of structure and purpose. 4. What are other inclusions found in bacteria? Be able to describe each type in terms of structure and purpose. 5. How do microcompartments differ from inclusions? What are specific examples? What is the composition and purpose? 6. What are anammoxosomes? What is their composition and purpose? 7. What are plasmids and what characteristics do they have? What are episomes? What is curing and what causes it? 8. What are bacterial endospores? What is their purpose? What characteristics do they have? What are the various layers of an endospore and what role does each layer play? Exploratory Questions (OPTIONAL) 1. What bacterial structures could be useful to scientists in addressing societal problems? 44 | Bacteria: Internal Components 6. Bacteria: Surface Structures Layers Outside the Cell Wall What have we learned so far, in terms of cell layers? All cells have a cell membrane. Most bacteria have a cell wall. But there are a couple of additional layers that bacteria may, or may not, have. These would be found outside of both the cell membrane and the cell wall, if present. Capsule A bacterial capsule is a polysaccharide layer that completely envelopes the cell. It is well organized and tightly packed, which explains its resistance to staining under the microscope. The capsule offers protection from a variety of different threats to the cell, such as desiccation, hydrophobic toxic materials (i.e. detergents), and bacterial viruses. The capsule can enhance the ability of bacterial pathogens to cause disease and can provide protection from phagocytosis (engulfment by white blood cells known as phagocytes). Lastly, it can help in attachment to surfaces. Slime Layer A bacterial slime layer is similar to the capsule in that it is typically composed of polysaccharides and it completely surrounds the cell. It also offers protection from various threats, such as desiccation and antibiotics. It can also allow for adherence to surfaces. So, how does it differ from the capsule? A slime layer is a loose, unorganized layer that is easily stripped from the cell that made it, as opposed to a capsule which integrates firmly around the bacterial cell wall. Bacteria: Surface Structures | 45 S-Layer Some bacteria have a highly organized layer made of secreted proteins or glycoproteins that self-assemble into a matrix on the outer part of the cell wall. This regularly structured S-layer is anchored into the cell wall, although it is not considered to be officially part of the cell wall in bacteria. S-layers have very important roles for the bacteria that have them, particularly in the areas of growth and survival, and cell integrity. S layers help maintain overall rigidity of the cell wall and surface layers, as well as cell shape, which are important for reproduction. S layers protect the cell from ion/pH changes, osmotic stress, detrimental enzymes, bacterial viruses, and predator bacteria. They can provide cell adhesion to other cells or surfaces. For pathogenic bacteria they can provide protection from phagocytosis. Structures Outside the Cell Wall Bacteria can also have structures outside of the cell wall, often bound to the cell wall and/or cell membrane. The building blocks for these structures are typically made within the cell and then secreted past the cell membrane and cell wall, to be assembled on the outside of the cell. Fimbriae (sing. fimbria) Fimbriae are thin filamentous appendages that extend from the cell, often in the tens or hundreds. They are composed of pilin proteins and are used by the cell to attach to surfaces. They can be particularly important for pathogenic bacteria, which use them to attach to host tissues. Pili (sing. pilus) Pili are very similar to fimbriae (some textbooks use the terms interchangeably) in that they are thin filamentous appendages that extend from the cell and are made of pilin proteins. Pili can be used for attachment as well, to both surfaces and host cells, such as the Neisseria gonorrhea cells that use their pili to grab onto sperm cells, for passage to the next human host. So, why would some researchers bother differentiating between fimbriae and pili? Pili are typically longer than fimbriae, with only 1-2 present on each cell, but that hardly seems enough to set the two structures apart. It really boils down to the fact that a few specific pili participate in functions beyond attachment. The conjugative pili participate in the process known as conjugation, which allows for the transfer of a small piece of DNA from a donor cell to a recipient cell. The type IV pili play a role in an unusual type of motility known as twitching motility, where a pilus attaches to a solid surface and then contracts, pulling the bacterium forward in a jerky motion. 46 | Bacteria: Surface Structures Flagella (sing. flagellum) Bacterial motility is typically provided by structures known as flagella. The bacterial flagellum differs in composition, structure, and mechanics from the eukaryotic flagellum, which operates as a flexible whip-like tail utilizing microtubules that are powered by ATP. The bacterial flagellum is rigid in nature, operates more like the propeller on a boat, and is powered by energy from the proton motive force. There are three main components to the bacterial flagellum: 1. the filament – a long thin appendage that extends from the cell surface. The filament is composed of the protein flagellin and is hollow. Flagellin proteins are transcribed in the cell cytoplasm and then transported across the cell membrane and cell wall. A bacterial flagellar filament grows from its tip (unlike the hair on your head), adding more and more flagellin units to extend the length until the correct size is reached. The flagellin units are guided into place by a protein cap. 2. the hook – this is a curved coupler that attaches the filament to the flagellar motor. 3. the motor – a rotary motor that spans both the cell membrane and the cell wall, with additional components for the gram negative outer membrane. The motor has two components: the basal body, which provides the rotation, and the stator, which provides the torque necessary for rotation to occur. The basal body consists of a central shaft surrounded by protein rings, two in the gram positive bacteria and four in the gram negative bacteria. The stator consists of Mot proteins that surround the ring(s) embedded within the cell membrane. Bacteria: Surface Structures | 47 Flagellum base diagram. By LadyofHats (Own work) [Public Domain], Via Wikipedia Commons Bacterial Movement Bacterial movement typically involves the use of flagella, although there are a few other possibilities as well (such as the use of type IV pili for twitching motility). But certainly the most common type of bacterial movement is swimming, which is accomplished with the use of a flagellum or flagella. 48 | Bacteria: Surface Structures Swimming Rotation of the flagellar basal body occurs due to the proton motive force, where protons that accumulate on the outside of the cell membrane are driven through pores in the Mot proteins, interacting with charges in the ring proteins as they pass across the membrane. The interaction causes the basal body to rotate and turns the filament extending from the cell. Rotation can occur at 200-1000 rpm and result in speeds of 60 cell lengths/second (for comparison, a cheetah moves at a maximum rate of 25 body lengths/second). Rotation can occur in a clockwise (CW) or a counterclockwise (CCW) direction, with different results to the cell. A bacterium will move forward, called a “run,” when there is a CCW rotation, and reorient randomly, called a “tumble,” when there is a CW rotation. Corkscrew Motility Some spiral-shaped bacteria, known as the Spirochetes, utilize a corkscrew-motility due to their unusual morphology and flagellar conformation. These gram negative bacteria have specialized flagella that attach to one end of the cell, extend back through the periplasm and then attach to the other end of the cell. When these endoflagella rotate they put torsion on the entire cell, resulting in a flexing motion that is particularly effective for burrowing through viscous liquids. Gliding Motility Gliding motility is just like it sounds, a slower and more graceful movement than the other forms covered so far. Gliding motility is exhibited by certain filamentous or bacillus bacteria and does not require the use of flagella. It does require that the cells be in contact with a solid surface, although more than one mechanism has been identified. Some cells rely on slime propulsion, where secreted slime propels the cell forward, where other cells rely on surface layer proteins to pull the cell forward. Chemotaxis Now that we have covered the basics of the bacterial flagellar motor and mechanics of bacterial swimming, let us combine the two topics to talk about chemotaxis or any other type of taxes (just not my taxes). Chemotaxis refers to the movement of an organism towards or away from a chemical. You can also have phototaxis, where an organism is responding to light. In chemotaxis, a favorable substance (such as a nutrient) is referred to as an attractant, while a substance with an adverse effect on the cell (such as a toxin) is referred to as a repellant. In the absence of either an attractant or a repellant a cell will engage in a “random walk,” where it alternates between tumbles and runs, in the end getting nowhere in particular. In the presence of a gradient of some type, the movements of the cell will become biased, resulting over Bacteria: Surface Structures | 49 time in the movement of the bacterium towards an attractant and away from any repellants. How does this happen? First, let us cover how a bacterium knows which direction to go. Bacteria rely on protein receptors embedded within their membrane, called chemoreceptors, which bind specific molecules. Binding typically results in methylation or phosphorylation of the chemoreceptor, which triggers an elaborate protein pathway that eventually impacts the rotation of the flagellar motor. The bacteria engage in temporal sensing, where they compare the concentration of a substance with the concentration obtained just a few seconds (or microseconds) earlier. In this way they gather information about the orientation of the concentration gradient of the substance. As a bacterium moves closer to the higher concentrations of an attractant, runs (dictated by CCW flagellar rotation) become longer, while tumbling (dictated by CW flagellar rotation) decreases. There will still be times that the bacterium will head off in the wrong direction away from an attractant since tumbling results in a random reorientation of the cell, but it won’t head in the wrong direction for very long. The resulting “biased random walk” allows the cell to quickly move up the gradient of an attractant (or move down the gradient of a repellant). Bacterial Movement. By Brudersohn (Own work (Original text: selbst erstellt)) [CC BY-SA 2.0 de], via Wikimedia Commons 50 | Bacteria: Surface Structures Key Words capsule, slime layer, S-layer, fimbriae/fimbria, pilin, pili/pilus, conjugative pili, conjugation, type IV pili, twitching motility, flagella/flagellum, filament, flagellin, hook, motor, basal body, stator, Mot proteins, swimming, clockwise (CW), counterclockwise (CCW), run, tumble, Spirochetes, corkscrew-motility, endoflagella, gliding motility, chemotaxis, phototaxis, attractant, repellant, random walk, chemoreceptors, temporal sensing, biased random walk. Study Questions 1. What are the compositions and functions of capsules and slime layers? When are they produced? How do capsules or slime layers increase the survival chances of bacteria in different environments? 2. What are fimbriae and pili; what are their compositions and functions? 3. What is the basic composition of a bacterial flagellum? What is the role of each component? How do bacterial flagella grow and how are proteins transported across the membrane? How are bacterial flagella attached to the body? How does the flagellar structure of gram negative bacteria differ from gram positive bacteria? 4. How does movement occur with a bacterial flagellum? Where does the energy come from? How does this information differ from flagella found in eukaryotes? 5. How do endoflagella differ from flagella and in what type of bacteria are they found? Where do they work better than flagella? 6. What is chemotaxis? How is information processed by a bacterium, to influence flagellar rotation? How does the direction of rotation of the flagella affect the way a bacterium moves? How long do stimuli last in chemotaxis and why is this important to the phenomenon? Exploratory Questions (OPTIONAL) 1. How could chemotaxis in microbes be used to address environmental pollution problems? Bacteria: Surface Structures | 51 7. Archaea The Archaea are a group of organisms that were originally thought to be bacteria (which explains the initial name of “archaeabacteria”), due to their physical similarities. More reliable genetic analysis revealed that the Archaea are distinct from both Bacteria and Eukaryotes, earning them their own domain in the Three Domain Classification originally proposed by Woese in 1977, alongside the Eukarya and the Bacteria. Phylogenetic Tree of Life. Similarities to Bacteria So, why were the archaea originally thought to be bacteria? Perhaps most importantly, they lack a nucleus or other membrane-bound organelles, putting them into the prokaryotic category (if you are using the traditional classification scheme). Most of them are unicellular, they have 70S sized ribosomes, they are typically a few micrometers in size, and they reproduce asexually only. They are known to have many of the same structures that bacteria can have, such as plasmids, inclusions, flagella, and pili. Capsules and slime layers have been found but appear to be rare in archaea. While archaea were originally isolated from extreme environments, such as places high in acid, salt, or heat, earning them the name “extremophiles,” they have more recently been isolated from all the places rich with bacteria: surface water, the ocean, human skin, soil, etc. 52 | Archaea Key Differences Plasma Membrane There are several characteristics of the plasma membrane that are unique to Archaea, setting them apart from other domains. One such characteristic is chirality of the glycerol linkage between the phopholipid head and the side chain. In archaea it is in the L-isomeric form, while bacteria and eukaryotes have the D-isomeric form. A second difference is the presence of an ether-linkage between the glycerol and the side chain, as opposed to the ester-linked lipids found in bacteria and eukaryotes. The ether-linkage provides more chemical stability to the membrane. A third and fourth difference are associated with the side chains themselves, unbranched fatty acids in bacteria and eukaryotes, while isoprenoid chains are found in archaea. These isoprenoid chains can have branching side chains. Archaea | 53 Comparison of Plasma Membrane Lipid Between Bacteria and Archaea. OpenStax, Structure of Prokaryotes. OpenStax CNX. Mar 28, 2014 http://cnx.org/contents/ 9e7c7540-5794-4c31-917d-fce7e50ea6dd@11. Lastly, the plasma membrane of Archaea can be found as monolayers, where the isoprene chains of one phospholipid connect with the isoprene chains of a phospholipid on the opposite side of the membrane. Bacteria and eukaryotes only have lipid bilayers, where the two sides of the membrane remain separated. Cell Wall Like bacteria, the archaeal cell wall is a semi-rigid structure designed to provide protection to the cell from the environment and from the internal cellular pressure. While the cell walls of bacteria typically contain 54 | Archaea peptidoglycan, that particular chemical is lacking in archaea. Instead, archaea display a wide variety of cell wall types, adapted for the environment of the organism. Some archaea lack a cell wall altogether. While it is not universal, a large number of Archaea have a proteinaceous S-layer that is considered to be part of the cell wall itself (unlike in Bacteria, where an S-layer is a structure in addition to the cell wall). For some Archaea the S-layer is the only cell wall component, while in others it is joined by additional ingredients (see below). The archaeal S-layer can be made of either protein or glycoprotein, often anchored into the plasma membrane of the cell. The proteins form a two-dimensional crystalline array with a smooth outer surface. A few S-layers are composed of two different S-layer proteins. While archaea lack peptidoglycan, a few contain a substance with a similar chemical structure, known as pseudomurein. Instead of NAM, it contains N-acetylalosaminuronic acid (NAT) linked to NAG, with peptide interbridges to increase strength. Methanochondroitin is a cell wall polymer found in some archaeal cells, similar in composition to the connective tissue component chondroitin, found in vertebrates. Some archaea have a protein sheath composed of a lattice structure similar to an S-layer. These cells are often found in filamentous chains, however, and the protein sheath encloses the entire chain, as opposed to individual cells. Archaea | 55 Cell Wall Structural Diversity. Ribosomes While archaea have ribosomes that are 70S in size, the same as bacteria, it was the rRNA nucleotide differences that provided scientists with the conclusive evidence to argue that archaea deserved a domain separate from the bacteria. In addition, archaeal ribosomes have a different shape than bacterial ribosomes, with proteins that are unique to archaea. This provides them with resistance to antibiotics that inhibit ribosomal function in bacteria. 56 | Archaea Structures Many of the structures found in bacteria have been discovered in archaea as well, although sometimes it is obvious that each structure was evolved independently, based on differences in substance and construction. Cannulae Cannulae, a structure unique to archaea, have been discovered in some marine archaeal strains. These hollow tube-like structures appear to connect cells after division, eventually leading to a dense network composed of numerous cells and tubes. This could serve as a means of anchoring a community of cells to a surface. Hami (sing. hamus) Another structure unique to archaea is the hamus, a long helical tube with three hooks at the far end. Hami appear to allow cells to attach both to one another and to surfaces, encouraging the formation of a community. Pili (sing. pilus) Pili have been observed in archaea, composed of proteins most likely modified from the bacterial pilin. The resulting tube-like structures have been shown to be used for attachment to surfaces. Flagella (sing. flagellum) The archaeal flagellum, while used for motility, differs so markedly from the bacterial flagellum that it has been proposed to call it an “archaellum,” to differentiate it from its bacterial counterpart. What is similar between the bacterial flagellum and the archaeal flagellum? Both are used for movement, where the cell is propelled by rotation of a rigid filament extending from the cell. After that the similarities end. What are the differences? The rotation of an archaeal flagellum is powered by ATP, as opposed to the proton motive force used in bacteria. The proteins making up the archaeal flagellum are similar to the proteins found in bacterial pili, rather than the bacterial flagellum. The archaeal flagellum filament is not hollow so growth occurs when flagellin proteins are inserted into the base of the filament, rather than being added to the end. The filament is made up of several different types of flagellin, while just one type Archaea | 57 is used for the bacterial flagellum filament. Clockwise rotation pushes an archaeal cells forward, while counterclockwise rotation pulls an archaeal cell backwards. An alternation of runs and tumbles is not observed. Classification Currently there are two recognized phyla of archaea: Euryarchaeota and Proteoarchaeota. Several additional phyla have been proposed (Nanoarchaeota, Korarchaeota, Aigarchaeota, Lokiarchaeota), but have yet to be officially recognized, largely due to the fact that the evidence comes from environmental sequences only. Key Words Archaea, L-isomeric form, D-isomeric form, ether-linkages, ester-linkages, isoprenoid chains, branching side chains, lipid monolayer, lipid bilayer, S-layer, pseudomurein, N-acetylalosaminuronic acid (NAT), methanochondroitin, protein sheath, cannulae, hamus/hami, pilus/pili, flagellum/flagella, archaellum, Euryarchaeota, Proteoarchaeota. Study Questions 1. How are the archaea similar to bacteria? 2. Describe the differences between the plasma membranes of archaea, compared to bacteria & eukaryotes. Explain the differences. 3. What types of cell walls exist in Archaea and what are they composed of? 4. How are archaeal ribosomes both similar and different from bacterial ribosomes? 5. How do the pili of archaea differ from those of bacteria? 6. What are cannulae and hami? What role could they play for archaea? 7. How does archaeal flagella differ from bacterial flagella, in terms of composition, assembly, and function? 8. Understand the commonalities and differences between archaea and bacteria, in term

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