Medical Microbiology: Chapter 2 - Cell Structure PDF

Naresuan University Access Pro...

Naresuan University Access Provided by: Jawetz, Melnick, & Adelberg's Medical Microbiology, 28e Chapter 2: Cell Structure INTRODUCTION This chapter discusses the basic structure and function of the components that make up eukaryotic and prokaryotic cells. It begins with a discussion of the microscope. Historically, the microscope first revealed the presence of bacteria and later the secrets of cell structure. Today, it remains a powerful tool in cell biology. OPTICAL METHODS The Light Microscope The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used. (Resolving power is the distance that must separate two point sources of light if they are to be seen as two distinct images.) With yellow light of a wavelength of 0.4 µm, the smallest separable diameters are thus about 0.2 µm (ie, one­third the width of a typical prokaryotic cell). The useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles. Several types of light microscopes, which are commonly used in microbiology, are discussed as follows. A. Bright­Field Microscope The bright­field microscope is the most commonly used in microbiology courses and consists of two series of lenses (objective and ocular lens), which function together to resolve the image. These microscopes generally employ a 100­power objective lens with a 10­power ocular lens, thus magnifying the specimen 1000 times. Particles 0.2 µm in diameter are therefore magnified to about 0.2 mm and so become clearly visible. Further magnification would give no greater resolution of detail and would reduce the visible area (field). With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium. Many bacteria are difficult to see well because of their lack of contrast with the surrounding medium. Dyes (stains) can be used to stain cells or their organelles and increase their contrast so that they can be more easily seen in the bright­field microscope. B. Phase­Contrast Microscope The phase­contrast microscope was developed to improve contrast differences between cells and the surrounding medium, making it possible to see living cells without staining them; with bright­field microscopes, killed and stained preparations must be used. The phase­contrast microscope takes advantage of the fact that light waves passing through transparent objects, such as cells, emerge in different phases depending on the properties of the materials through which they pass. This effect is amplified by a special ring in the objective lens of a phase­contrast microscope, leading to the formation of a dark image on a light background (Figure 2­1). FIGURE 2­1 Using the phase contrast illumination technique, this photomicrograph of a wet mount of a vaginal discharge specimen revealed the presence of the flagellated protozoan, Trichomonas vaginalis. (Courtesy of Centers for Disease Control and Prevention, Public Health Image Library, ID# 5238.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 1 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility FIGURE 2­1 Naresuan University Access Provided by: Using the phase contrast illumination technique, this photomicrograph of a wet mount of a vaginal discharge specimen revealed the presence of the flagellated protozoan, Trichomonas vaginalis. (Courtesy of Centers for Disease Control and Prevention, Public Health Image Library, ID# 5238.) C. Dark­Field Microscope The dark­field microscope is a light microscope in which the lighting system has been modified to reach the specimen from the sides only. This is accomplished through the use of a special condenser that both blocks direct light rays and deflects light off a mirror on the side of the condenser at an oblique angle. This creates a “dark field” that contrasts against the highlighted edge of the specimens and results when the oblique rays are reflected from the edge of the specimen upward into the objective of the microscope. Resolution by dark­field microscopy is quite high. Thus, this technique has been particularly useful for observing organisms such as Treponema pallidum, a spirochete that is smaller than 0.2 µm in diameter and therefore cannot be observed with a bright­field or phase­contrast microscope (Figure 2­2A). FIGURE 2­2 A : Positive dark­field examination. Treponemes are recognizable by their characteristic corkscrew shape and deliberate forward and backward movement with rotation about the longitudinal axis. (Reproduced with permission. © Charles Stratton/Visuals Unlimited.) B : Fluorescence photomicrograph. A rod­shaped bacterium tagged with a fluorescent marker. (© Evans Roberts.) C : Scanning electron microscope of bacteria —Staphylococcus aureus (32,000×). (Reproduced with permission from David M. Phillips/Photo Researchers, Inc.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 2 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility A : Positive dark­field examination. Treponemes are recognizable by their characteristic corkscrew shape and deliberate forward and backward Naresuan University movement with rotation about the longitudinal axis. (Reproduced with permission. © Charles Stratton/Visuals Unlimited.) B : Fluorescence Access Provided by: photomicrograph. A rod­shaped bacterium tagged with a fluorescent marker. (© Evans Roberts.) C : Scanning electron microscope of bacteria —Staphylococcus aureus (32,000×). (Reproduced with permission from David M. Phillips/Photo Researchers, Inc.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 3 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility D. Fluorescence Microscope  Naresuan University Access Provided by: D. Fluorescence Microscope The fluorescence microscope is used to visualize specimens that fluoresce, which is the ability to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). Some organisms fluoresce naturally because of the presence within the cells of naturally fluorescent substances such as chlorophyll. Those that do not naturally fluoresce may be stained with a group of fluorescent dyes called fluorochromes. Fluorescence microscopy is widely used in clinical diagnostic microbiology. For example, the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by the cell envelope of Mycobacterium tuberculosis, the bacterium that causes tuberculosis. When the dye is applied to a specimen suspected of containing M. tuberculosis and exposed to ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms against a dark background. The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent­antibody (FA) technique or immunofluorescence. In this technique, specific antibodies (eg, antibodies to Legionella pneumophila) are chemically labeled with a fluorochrome such as fluorescein isothiocyanate (FITC). These fluorescent antibodies are then added to a microscope slide containing a clinical specimen. If the specimen contains L. pneumophila, the fluorescent antibodies will bind to antigens on the surface of the bacterium, causing it to fluoresce when exposed to ultraviolet light (Figure 2­2B). E. Differential Interference Contrast Microscope Differential interference contrast (DIC) microscopes employ a polarizer to produce polarized light. The polarized light beam passes through a prism that generates two distinct beams; these beams pass through the specimen and enter the objective lens, where they are recombined into a single beam. Because of slight differences in refractive index of the substances each beam passed through, the combined beams are not totally in phase but instead create an interference effect, which intensifies subtle differences in cell structure. Structures, such as spores, vacuoles, and granules, appear three dimensional. DIC microscopy is particularly useful for observing unstained cells because of its ability to generate images that reveal internal cell structures that are less apparent by bright­field techniques. The Electron Microscope The high resolving power of electron microscopes has enabled scientists to observe the detailed structures of prokaryotic and eukaryotic cells. The superior resolution of the electron microscope is because electrons have a much shorter wavelength than the photons of white light. There are two types of electron microscopes in general use: The transmission electron microscope (TEM), which has many features in common with the light microscope; and the scanning electron microscope (SEM). The TEM was the first to be developed and uses a beam of electrons projected from an electron gun and directed or focused by an electromagnetic condenser lens onto a thin specimen. As the electrons strike the specimen, they are differentially scattered by the number and mass of atoms in the specimen; some electrons pass through the specimen and are gathered and focused by an electromagnetic objective lens, which presents an image of the specimen to the projector lens system for further enlargement. The image is visualized by allowing it to impinge on a screen that fluoresces when struck with the electrons. The image can be recorded Downloaded 2024­8­4 on photographic film. TEM 12:7 A resolve can Your IPparticles is 110.170.245.47 0.001 µm apart. Thus, viruses with diameters of 0.01–0.2 µm are easily resolved by TEM. Chapter 2: Cell Structure, Page 4 / 44 ©2024 The SEMMcGraw generallyHill. hasAlla Rights Reserved. lower resolving Terms power thanofthe Use Privacy TEM; Policy however, it is particularly Notice Accessibility useful for providing three­dimensional images of the surface of microscopic objects. Electrons are focused by means of lenses into a very fine point. The interaction of electrons with the specimen results in the release of different forms of radiation (eg, secondary electrons) from the surface of the material, which can be captured by an appropriate detector, There are two types of electron microscopes in general use: The transmission electron microscope (TEM), which has many features in common Naresuan with the light microscope; and the scanning electron microscope (SEM). The TEM was the first to be developed and uses a beam University of electrons projected from an electron gun and directed or focused by an electromagnetic condenser lens onto a thin specimen. As the electrons strike Access theby: Provided specimen, they are differentially scattered by the number and mass of atoms in the specimen; some electrons pass through the specimen and are gathered and focused by an electromagnetic objective lens, which presents an image of the specimen to the projector lens system for further enlargement. The image is visualized by allowing it to impinge on a screen that fluoresces when struck with the electrons. The image can be recorded on photographic film. TEM can resolve particles 0.001 µm apart. Thus, viruses with diameters of 0.01–0.2 µm are easily resolved by TEM. The SEM generally has a lower resolving power than the TEM; however, it is particularly useful for providing three­dimensional images of the surface of microscopic objects. Electrons are focused by means of lenses into a very fine point. The interaction of electrons with the specimen results in the release of different forms of radiation (eg, secondary electrons) from the surface of the material, which can be captured by an appropriate detector, amplified, and then imaged on a television screen (Figure 2­2C). An important technique in electron microscopy is the use of “shadowing.” This involves depositing a thin layer of heavy metal (eg, platinum) on the specimen by placing it in the path of a beam of metal ions in a vacuum. The beam is directed at a low angle to the specimen so that it acquires a “shadow” in the form of an uncoated area on the other side. When an electron beam is then passed through the coated preparation in the electron microscope and a positive print is made from the “negative” image, a three­dimensional effect is achieved (eg, see Figure 2­24). Other important techniques in electron microscopy include the use of ultrathin sections of embedded material, a method of freeze­drying specimens that prevents the distortion caused by conventional drying procedures, and the use of negative staining with an electron­dense material such as phosphotungstic acid or uranyl salts (eg, see Figure 42­1). Without these heavy metal salts, there would not be enough contrast to detect the details of the specimen. Confocal Scanning Laser Microscope The confocal scanning laser microscope (CSLM) couples a laser light source to a light microscope. In confocal scanning laser microscopy, a laser beam is bounced off a mirror that directs the beam through a scanning device. Then the laser beam is directed through a pinhole that precisely adjusts the plane of focus of the beam to a given vertical layer within the specimen. By precisely illuminating only a single plane of the specimen, illumination intensity drops off rapidly above and below the plane of focus, and stray light from other planes of focus are minimized. Thus, in a relatively thick specimen, various layers can be observed by adjusting the plane of focus of the laser beam. Cells are often stained with fluorescent dyes to make them more visible. Alternatively, false color images can be generated by adjusting the microscope in such a way as to make different layers take on different colors. The CSLM is equipped with computer software to assemble digital images for subsequent image processing. Thus, images obtained from different layers can be stored and then digitally overlaid to reconstruct a three­ dimensional image of the entire specimen (Figure 2­3). FIGURE 2­3 Using laser light, CDC laboratory scientists sometimes work with a confocal microscope when studying various pathogens. (Courtesy of James Gathany, Centers for Disease Control and Prevention, Public Health Image Library, ID# 1960.) Downloaded Scanning Probe2024­8­4 12:7 A Your IP is 110.170.245.47 Microscopes Chapter 2: Cell Structure, Page 5 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility A new class of microscopes, called scanning probe microscopes, measures surface features by moving a sharp probe over the object’s surface. The scanning tunneling microscope and the atomic force microscope are the examples of this new class of microscopes, which enable scientists to  Naresuan University Access Provided by: Scanning Probe Microscopes A new class of microscopes, called scanning probe microscopes, measures surface features by moving a sharp probe over the object’s surface. The scanning tunneling microscope and the atomic force microscope are the examples of this new class of microscopes, which enable scientists to view atoms or molecules on the surface of a specimen. For example, interactions between proteins of the bacterium Escherichia coli can be studied with the atomic force microscope (Figure 2­4). FIGURE 2­4 Atomic force microscopy. Micrograph of a fragment of DNA. The bright peaks are enzymes attached to the DNA. (Reproduced with permission from Torunn Berg, Photo Researchers, Inc.) EUKARYOTIC CELL STRUCTURE The Nucleus The nucleus contains the cell’s genome. It is bounded by a membrane, which is composed of two lipid bilayer membranes: the inner and the outer membrane. The inner membrane is usually a simple sac, but the outermost membrane is, in many places, continuous with the endoplasmic reticulum (ER). The nuclear membrane exhibits selective permeability because of pores, which consist of a complex of several proteins whose function is to import substances into and export substances out of the nucleus. The chromosomes of eukaryotic cells contain linear DNA macromolecules arranged as a double helix. They are only visible with a light microscope when the cell is undergoing division and the DNA is in a highly condensed form; at other times, the chromosomes are not condensed and appear as in Figure 2­5. Eukaryotic DNA macromolecules are associated with basic proteins called histones that bind to the DNA by ionic interactions. FIGURE 2­5 Eukaryotic cells. A : Diagrammatic representation of an animal cell. B : Diagrammatic representation of a plant cell. C : Micrograph of an animal cell shows several membrane­bound structures, including mitochondria and a nucleus. (Fig. 2­3(A) and (B) Reproduced with permission from Nester EW, Anderson DG, Roberts CE, et al: Microbiology: A Human Perspective, 6th ed. McGraw­Hill, 2009. © McGraw­Hill Education. Fig. 2­3(C) Reproduced with permission from Thomas Fritsche, MD, PhD.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 6 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility Eukaryotic cells. A : Diagrammatic representation of an animal cell. B : Diagrammatic representation of a plant cell. C : Micrograph of an animal cell Naresuan University shows several membrane­bound structures, including mitochondria and a nucleus. (Fig. 2­3(A) and (B) Reproduced with permission from Nester EW, Access Provided by: Anderson DG, Roberts CE, et al: Microbiology: A Human Perspective, 6th ed. McGraw­Hill, 2009. © McGraw­Hill Education. Fig. 2­3(C) Reproduced with permission from Thomas Fritsche, MD, PhD.) A structure often visible within the nucleus is the nucleolus, an area rich in RNA that is the site of ribosomal RNA synthesis (see Figure 2­5). Ribosomal proteins synthesized in the cytoplasm are transported into the nucleolus and combine with ribosomal RNA to form the small and large subunits of the eukaryotic ribosome. These are then exported to the cytoplasm, where they associate to form an intact ribosome that can function in protein synthesis. Cytoplasmic Structures The cytoplasm of eukaryotic cells is characterized by the presence of an ER, vacuoles, self­reproducing plastids, and an elaborate cytoskeleton composed of microtubules, microfilaments, and intermediate filaments. The endoplasmic reticulum (ER) is a network of membrane­bound channels continuous with the nuclear membrane. Two types of ER are recognized: rough, to which 80S ribosomes are attached; and smooth, which does not have attached ribosomes (see Figure 2­5). Rough ER is a major producer of glycoproteins as well as new membrane material that is transported throughout the cell; smooth ER participates in the synthesis of lipids and in some aspects of carbohydrate metabolism. The Golgi complex consists of a stack of membranes that function in concert with the ER to chemically modify and sort products of the ER into those destined to be secreted and those that function in other membranous structures of the cell. The plastids include mitochondria and chloroplasts. Several lines of evidence suggest that mitochondria and chloroplasts arose from the engulfment of a prokaryotic cell by a larger cell (endosymbiosis). Current hypotheses, making use of mitochondrial genome and proteome data, suggest that the mitochondrial ancestor was most closely related to Alphaproteobacteria and that chloroplasts are related to nitrogen­fixing cyanobacteria. Mitochondria are of prokaryotic size (Figure 2­5), and its membrane, which lacks sterols, is much less rigid than the eukaryotic cell’s cytoplasmic membrane, which does contain sterols. Mitochondria contain two sets of membranes. The outermost membrane is rather permeable, having numerous minute channels that allow passage of ions and small molecules (eg, adenosine triphosphate [ATP]). Invagination of the outer membrane forms a system of inner folded membranes called cristae. The cristae are the sites of enzymes involved in respiration and ATP production. Cristae also contain specific transport proteins that regulate passage of metabolites into and out of the mitochondrial matrix. The matrix contains a number of enzymes, particularly those of the citric acid cycle. Chloroplasts are the photosynthetic cell organelles that can convert the energy of sunlight into chemical energy through photosynthesis. Chlorophyll and all other components needed for photosynthesis are located in a series of flattened membrane discs called thylakoids. The size, shape, and number of chloroplasts per cell vary markedly; in contrast to mitochondria, chloroplasts are generally much larger than prokaryotes. Mitochondria and chloroplasts contain their own DNA, which exists in a covalently closed circular form and codes for some (not all) of their constituent proteins and transfer RNAs. Mitochondria and chloroplasts also contain 70S ribosomes, the same as those of prokaryotes. Eukaryotic microorganisms that were previously thought to lack mitochondria (amitochondriate eukaryotes) have been recently shown to contain some mitochondrial remnants either through the maintenance of membrane­enclosed respiratory organelles called hydrogenosomes, mitosomes, or nuclear genes of mitochondrial origin. There are two types of amitochondriate eukaryotes: type II (eg, Trichomonas vaginalis) harbors a hydrogenosome, while type I (eg, Giardia lamblia) lacks organelles involved in core energy metabolism. Some amitochondrial parasites (eg, Downloaded 2024­8­4) are Entamoeba histolytica 12:7intermediate A Your IP isand 110.170.245.47 appear to be evolving from a type II to type I. Some hydrogenosomes have been identified that contain Chapter 2: Cell Structure, Page 7 / 44 DNA ©2024andMcGraw ribosomes. Hill. The hydrogenosome, All Rights Reserved. although Terms ofsimilar Use in size toPolicy Privacy mitochondria, Notice lacks cristae and the enzymes of the tricarboxylic acid cycle. Accessibility Pyruvate is taken up by the hydrogenosome, and H2, CO2, acetate, and ATP are produced. The mitosome has only recently been discovered and named, and its function has not been well characterized. the same as those of prokaryotes. Naresuan University Eukaryotic microorganisms that were previously thought to lack mitochondria (amitochondriate eukaryotes) have been recently shown Access to contain Provided by: some mitochondrial remnants either through the maintenance of membrane­enclosed respiratory organelles called hydrogenosomes, mitosomes, or nuclear genes of mitochondrial origin. There are two types of amitochondriate eukaryotes: type II (eg, Trichomonas vaginalis) harbors a hydrogenosome, while type I (eg, Giardia lamblia) lacks organelles involved in core energy metabolism. Some amitochondrial parasites (eg, Entamoeba histolytica) are intermediate and appear to be evolving from a type II to type I. Some hydrogenosomes have been identified that contain DNA and ribosomes. The hydrogenosome, although similar in size to mitochondria, lacks cristae and the enzymes of the tricarboxylic acid cycle. Pyruvate is taken up by the hydrogenosome, and H2, CO2, acetate, and ATP are produced. The mitosome has only recently been discovered and named, and its function has not been well characterized. Lysosomes are membrane­enclosed vesicles that contain various digestive enzymes that the cell uses to digest macromolecules such as proteins, fats, and polysaccharides. The lysosome allows these enzymes to be partitioned away from the cytoplasm proper, where they could destroy key cellular macromolecules if not contained. After the hydrolysis of macromolecules in the lysosome, the resulting monomers pass from the lysosome into the cytoplasm, where they serve as nutrients. The peroxisome is a membrane­enclosed structure whose function is to produce H2O2 from the reduction of O2 by various hydrogen donors. The H2O2 produced in the peroxisome is subsequently degraded to H2O and O2 by the enzyme catalase. Peroxisomes are believed to be of evolutionary origin unrelated to mitochondria. The cytoskeleton is a three­dimensional structure that fills the cytoplasm. Eukaryotic cells contain three main kinds of cytoskeletal filaments: microfilaments, intermediate filaments, and microtubules. Each cytoskeletal filament type is formed by polymerization of a distinct type of protein subunit and has its own shape and intracellular distribution. Microfilaments are about 7 nm in diameter and are polymers composed of the protein actin. These fibers form scaffolds throughout the cell, defining and maintaining the shape of the cell. Microfilaments can also carry out intracellular transport/trafficking, and cellular movements, including gliding, contraction, and cytokinesis. Microtubules are hollow cylinders about 23 nm in diameter (lumen is approximately 15 nm in diameter) most commonly comprising 13 protofilaments that, in turn, are polymers of alpha and beta tubulin. Microtubules assist microfilaments in maintaining cell structure, form the spindle fibers for separating chromosomes during mitosis, and play an important role in cell motility. Intermediate filaments are composed of various proteins (eg, keratin, lamin, and desmin) depending on the type of cell in which they are found. They are normally 8–12 nm in diameter and provide tensile strength for the cell. They are most commonly known as the support system or “scaffolding” for the cell and nucleus. All filaments react with accessory proteins (eg, Rho and dynein) that regulate and link the filaments to other cell components and each other. Surface Layers The cytoplasm is enclosed within a plasma membrane composed of protein and phospholipid similar to the prokaryotic cell membrane illustrated later (see Figure 2­13). Most animal cells have no other surface layers; however, plant cells have an outer cell wall composed of cellulose (Figure 2­5B). Many eukaryotic microorganisms also have an outer cell wall, which may be composed of a polysaccharide such as cellulose or chitin or may be inorganic (eg, the silica wall of diatoms). Motility Organelles Many eukaryotic microorganisms have organelles called flagella (eg, T. vaginalis) or cilia (eg, Paramecium) that move with a wavelike motion to propel the cell through water. Eukaryotic flagella emanate from the polar region of the cell, and cilia, which are shorter than flagella, surround the cell (Figure 2­6). Both the flagella and the cilia of eukaryotic cells have the same basic structure and biochemical composition. Both consist of a series of microtubules, hollow protein cylinders composed of a protein called tubulin surrounded by a membrane. The arrangement of the microtubules is commonly referred to as the “9 + 2 arrangement” because it consists of nine doublets of microtubules surrounding two single central microtubules (Figure 2­7). Each doublet is connected to another by the protein dynein. The dynein arms attached to the microtubule function as the molecular motors. FIGURE 2­6 A paramecium moves with the aid of cilia on the cell surface. (© Manfred Kage.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 8 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility motors. Naresuan University FIGURE 2­6 Access Provided by: A paramecium moves with the aid of cilia on the cell surface. (© Manfred Kage.) FIGURE 2­7 Cilia and flagella structure. A : An electron micrograph of a cilium cross section. Note the two central microtubles surrounded by nine microtubule doublets (160,000×). (Reproduced with permission. © Kallista Images/Visuals Unlimited, Inc.) B : A diagram of cilia and flagella structure. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw­Hill; 2008. © McGraw­Hill Education.) PROKARYOTIC CELL STRUCTURE The prokaryotic cell is simpler than the eukaryotic cell at every level, with one exception: The cell envelope is more complex. The Nucleoid Prokaryotes have no true nuclei; instead they package their DNA in a structure known as the nucleoid. The negatively charged DNA is at least partially neutralized by small polyamines and magnesium ions. Nucleoid­associated proteins exist in bacteria and are distinct from histones in eukaryotic chromatin. Electron micrographs of a typical prokaryotic cell reveal the absence of a nuclear membrane and a mitotic apparatus. The exception to this rule is the planctomycetes, a divergent group of aquatic bacteria, which have a nucleoid surrounded by a nuclear envelope consisting of two membranes. The distinction between prokaryotes and eukaryotes that still holds is that prokaryotes have no eukaryotic­type mitotic apparatus. The nuclear region is filled with DNA fibrils (Figure 2­8). The nucleoid of most bacterial cells consists of a single continuous circular molecule ranging in size from 0.58 to almost 10 million Downloaded base pairs. 2024­8­4 However, 12:7 A Your IPaisfew bacteria have been shown to have two, three, or even four dissimilar chromosomes. For example, Vibrio 110.170.245.47 cholerae2: Chapter and Brucella Cell melitensis have two dissimilar chromosomes. There are exceptions to this rule of circularity because some prokaryotes Structure, Page(eg, 9 / 44 ©2024 McGraw Borrelia Hill. and burgdorferi All Rights Reserved. Streptomyces Terms) have coelicolor of Use Privacy been shownPolicy to have Notice a linear chromosome. Accessibility FIGURE 2­8  Naresuan Electron micrographs of a typical prokaryotic cell reveal the absence of a nuclear membrane and a mitotic apparatus. The exception to this ruleUniversity is the Access Provided by: planctomycetes, a divergent group of aquatic bacteria, which have a nucleoid surrounded by a nuclear envelope consisting of two membranes. The distinction between prokaryotes and eukaryotes that still holds is that prokaryotes have no eukaryotic­type mitotic apparatus. The nuclear region is filled with DNA fibrils (Figure 2­8). The nucleoid of most bacterial cells consists of a single continuous circular molecule ranging in size from 0.58 to almost 10 million base pairs. However, a few bacteria have been shown to have two, three, or even four dissimilar chromosomes. For example, Vibrio cholerae and Brucella melitensis have two dissimilar chromosomes. There are exceptions to this rule of circularity because some prokaryotes (eg, Borrelia burgdorferi and Streptomyces coelicolor) have been shown to have a linear chromosome. FIGURE 2­8 The nucleoid. A : Color­enhanced transmission electron micrograph of E. coli with the DNA shown in red. (Reproduced with permission. © CNRI/SPL/Photo Researchers, Inc.) B : Chromosome released from a gently lysed cell of E. coli. Note how tightly packaged the DNA must be inside the bacterium. (Reproduced with permission. © Dr. Gopal Murti/SPL/Photo Researchers Inc.) In bacteria, the number of nucleoids, and therefore the number of chromosomes, depends on the growth conditions. Rapidly growing bacteria have more nucleoids per cell than slowly growing ones; however, when multiple copies are present, they are all the same (ie, prokaryotic cells are haploid). Cytoplasmic Structures Prokaryotic cells lack autonomous plastids, such as mitochondria and chloroplasts; the electron transport enzymes are localized instead in the cytoplasmic membrane. The photosynthetic pigments (carotenoids, bacteriochlorophyll) of photosynthetic bacteria are contained in intracytoplasmic membrane systems of various morphologies. Membrane vesicles (chromatophores) or lamellae are commonly observed membrane types. Some photosynthetic bacteria have specialized nonunit membrane­enclosed structures called chlorosomes. In some cyanobacteria (formerly known as Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 blue­green Chapter 2: algae), the photosynthetic membranes often form multilayered structures known as thylakoids (Figure 2­9). The major accessory Cell Structure, Page 10 / 44 ©2024 McGraw pigments used forHill. Allharvesting light Rights Reserved. Terms of Use are the phycobilins Privacy found Policysurface on the outer Notice of the Accessibility thylakoid membranes. FIGURE 2­9 Cytoplasmic Structures Naresuan University Prokaryotic cells lack autonomous plastids, such as mitochondria and chloroplasts; the electron transport enzymes are localized instead in theby: Access Provided cytoplasmic membrane. The photosynthetic pigments (carotenoids, bacteriochlorophyll) of photosynthetic bacteria are contained in intracytoplasmic membrane systems of various morphologies. Membrane vesicles (chromatophores) or lamellae are commonly observed membrane types. Some photosynthetic bacteria have specialized nonunit membrane­enclosed structures called chlorosomes. In some cyanobacteria (formerly known as blue­green algae), the photosynthetic membranes often form multilayered structures known as thylakoids (Figure 2­9). The major accessory pigments used for light harvesting are the phycobilins found on the outer surface of the thylakoid membranes. FIGURE 2­9 Thin section of Synechocystis during division. Many structures are visible. (Reproduced from Stanier RY: The position of cyanobacteria in the world of phototrophs. Carlsberg Res Commun 42:77­98, 1977. With kind permission of Springer + Business Media.) Bacteria often store reserve materials in the form of insoluble granules, which appear as refractile bodies in the cytoplasm when viewed by phase­ contrast microscopy. These so­called inclusion bodies almost always function in the storage of energy or as a reservoir of structural building blocks. Most cellular inclusions are bounded by a thin nonunit membrane consisting of lipid, which serves to separate the inclusion from the cytoplasm proper. One of the most common inclusion bodies consists of poly­β­hydroxybutyric acid (PHB), a lipid­like compound consisting of chains of β­ hydroxybutyric acid units connected through ester linkages. PHB is produced when the source of nitrogen, sulfur, or phosphorous is limited and there is excess carbon in the medium (Figure 2­10A). Another storage product formed by prokaryotes when carbon is in excess is glycogen, which is a polymer of glucose. PHB and glycogen are used as carbon sources when protein and nucleic acid synthesis are resumed. A variety of prokaryotes are capable of oxidizing reduced sulfur compounds, such as hydrogen sulfide and thiosulfate, producing intracellular granules of elemental sulfur (Figure 2­10B). As the reduced sulfur source becomes limiting, the sulfur in the granules is oxidized, usually to sulfate, and the granules slowly disappear. Many bacteria accumulate large reserves of inorganic phosphate in the form of granules of polyphosphate. These granules can be degraded and used as sources of phosphate for nucleic acid and phospholipid synthesis to support growth. These granules are sometimes termed volutin granules or metachromatic granules because they stain red with a blue dye. They are characteristic features of Corynebacterium (see Chapter 13). FIGURE 2­10 Inclusion bodies in bacteria. A : Electron micrograph of B. megaterium (30,500×) showing poly­β­hydroxybutyric acid inclusion body, PHB; cell wall, CW; nucleoid, N; plasma Downloaded 2024­8­4membrane, PM; “mesosome,” 12:7 A Your M; and ribosomes, R. (Reproduced with permission. © Ralph A. Slepecky/Visuals Unlimited.) B : IP is 110.170.245.47 Cromatium Chapter vinosum 2: Cell , a purple sulfur bacterium, with intracellular sulfur granules, bright field microscopy (2000×). (Reproduced with permission Structure, Page 11from / 44 ©2024 Holt McGraw J (editor): Hill. The All Rights Shorter Reserved. Bergey’s Manual Terms of Use Privacy of Determinative Policy, 8th Bacteriology Notice Accessibility ed. Williams & Wilkins, 1977. Copyright Bergey’s Manual Trust.) Chapter 13). Naresuan University Access Provided by: FIGURE 2­10 Inclusion bodies in bacteria. A : Electron micrograph of B. megaterium (30,500×) showing poly­β­hydroxybutyric acid inclusion body, PHB; cell wall, CW; nucleoid, N; plasma membrane, PM; “mesosome,” M; and ribosomes, R. (Reproduced with permission. © Ralph A. Slepecky/Visuals Unlimited.) B : Cromatium vinosum, a purple sulfur bacterium, with intracellular sulfur granules, bright field microscopy (2000×). (Reproduced with permission from Holt J (editor): The Shorter Bergey’s Manual of Determinative Bacteriology, 8th ed. Williams & Wilkins, 1977. Copyright Bergey’s Manual Trust.) Certain groups of autotrophic bacteria that fix carbon dioxide to make their biochemical building blocks contain polyhedral bodies surrounded by a protein shell (carboxysomes) containing the key enzyme of CO2 fixation, ribulosebisphosphate carboxylase (see Figure 2­9). Magnetosomes are intracellular crystal particles of the iron mineral magnetite (Fe3O4) that allow certain aquatic bacteria to exhibit magnetotaxis (ie, migration or orientation of the cell with respect to the earth’s magnetic field). Magnetosomes are surrounded by a nonunit membrane containing phospholipids, proteins, and glycoproteins. Gas vesicles are found almost exclusively in microorganisms from aquatic habitats, where they provide buoyancy. The gas vesicle membrane is a 2­nm­thick layer of protein, impermeable to water and solutes but permeable to gases; thus, gas vesicles exist as gas­filled structures surrounded by the constituents of the cytoplasm (Figure 2­11). FIGURE 2­11 Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Transverse Chapter section 2: Cell of a dividing cell of the cyanobacterium Microcystis species showing hexagonal stacking of the cylindric gas vesicles (31,500×). Structure, Page 12 / 44 ©2024 McGraw (Micrograph by HSHill. All Rights Pankratz. Reserved.with Reproduced Terms of Use from permission Privacy Policy Walsby AE: Gas Notice Accessibility vesicles. Microbiol Rev 1994;58:94.) proteins, and glycoproteins. Gas vesicles are found almost exclusively in microorganisms from aquatic habitats, where they provide buoyancy. The Naresuan University gas vesicle membrane is a 2­nm­thick layer of protein, impermeable to water and solutes but permeable to gases; thus, gas vesicles exist as gas­filled Access Provided by: structures surrounded by the constituents of the cytoplasm (Figure 2­11). FIGURE 2­11 Transverse section of a dividing cell of the cyanobacterium Microcystis species showing hexagonal stacking of the cylindric gas vesicles (31,500×). (Micrograph by HS Pankratz. Reproduced with permission from Walsby AE: Gas vesicles. Microbiol Rev 1994;58:94.) The most numerous intracellular structure in most bacteria is the ribosome, the site of protein synthesis in all living organisms. All prokaryotes have 70S ribosomes, while eukaryotes contain larger 80S ribosomes in their cytoplasm. The 70S ribosome is made up of 50S and 30S subunits. The 50S subunit contains the 23S and 5S ribosomal RNA (rRNA), while the 30S subunit contains the 16S rRNA. These rRNA molecules are complexed with a large number of ribosomal proteins. The bacterial cytoplasm also contains homologs of all the major cytoskeletal proteins of eukaryotic cells as well as additional proteins that play cytoskeletal roles (Figure 2­12). Actin homologs (eg, MreB and Mbl) perform a variety of functions, helping to determine cell shape, segregate chromosomes, and localize proteins within the cell. Nonactin homologs (eg, FtsZ) and unique bacterial cytoskeletal proteins (eg, SecY and MinD) are involved in determining cell shape and in regulation of cell division and chromosome segregation. FIGURE 2­12 The prokaryotic cytoskeleton. Visualization of the MreB­like cytoskeletal protein (Mbl) of B. subtilis. The Mbl protein has been fused with green fluorescent protein, and live cells have been examined by fluorescence microscopy. A : Arrows point to the helical cytoskeleton cables that extend the length of the cells. B : Three of the cells from A are shown at a higher magnification. (Courtesy of Rut Carballido­Lopez and Jeff Errington.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 13 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility FIGURE 2­12 Naresuan University The prokaryotic cytoskeleton. Visualization of the MreB­like cytoskeletal protein (Mbl) of B. subtilis. The Mbl protein has been fused with green Access Provided by: fluorescent protein, and live cells have been examined by fluorescence microscopy. A : Arrows point to the helical cytoskeleton cables that extend the length of the cells. B : Three of the cells from A are shown at a higher magnification. (Courtesy of Rut Carballido­Lopez and Jeff Errington.) The Cell Envelope Prokaryotic cells are surrounded by complex envelope layers that differ in composition among the major groups. These structures protect the organisms from hostile environments, such as extreme osmolarity, harsh chemicals, and even antibiotics. The Plasma Membrane A. Structure The plasma membrane, also called the bacterial cytoplasmic membrane, is visible in electron micrographs of thin sections (see Figure 2­9). It is a typical “unit membrane” composed of phospholipids and upward of 200 different proteins. Proteins account for approximately 70% of the mass of the membrane, which is a considerably higher proportion than that of mammalian cell membranes. Figure 2­13 illustrates a model of membrane organization. The membranes of prokaryotes are distinguished from those of eukaryotic cells by the absence of sterols (with some exceptions, eg, mycoplasmas, which also lack a cell wall, incorporate sterols, such as cholesterol, into their membranes when growing in sterol­containing media). However, many bacteria contain structurally related compounds called hopanoids, which likely fulfill the same function. Unlike eukaryotes, bacteria can have a wide variety of fatty acids within their membranes. Along with the typical saturated and unsaturated fatty acids, bacterial membranes can contain fatty acids with additional methyl, hydroxy, or cyclic groups. The relative proportions of these fatty acids can be modulated by the bacterium to maintain the optimum fluidity of the membrane. For example, at least 50% of the cytoplasmic membrane must be in the semifluid state for cell growth to occur. At low temperatures, this is achieved by greatly increased synthesis and incorporation of unsaturated fatty acids into the phospholipids of the cell membrane. FIGURE 2­13 Bacterial plasma membrane structure. This diagram of the fluid mosaic model of bacterial membrane structure shown the integral proteins (green and red) floating in a lipid bilayer. Peripheral proteins (yellow) are associated loosely with the inner membrane surface. Small spheres represent the hydrophilic ends of membrane phospholipids and wiggly tails, the hydrophobic fatty acid chains. Other membrane lipids such as hopanoids (purple) may be present. For the sake of clarity, phospholipids are shown proportionately much larger size than in real membranes. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw­Hill; 2008. © McGraw­Hill Education.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 14 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility hydrophilic ends of membrane phospholipids and wiggly tails, the hydrophobic fatty acid chains. Other membrane lipids such as hopanoids (purple) Naresuan University may be present. For the sake of clarity, phospholipids are shown proportionately much larger size than in real membranes. (Reproduced with Access Provided by: permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw­Hill; 2008. © McGraw­Hill Education.) The cell membranes of the Archaea (see Chapter 1) differ from those of the Bacteria. Some Archaeal cell membranes contain unique lipids, isoprenoids, rather than fatty acids, linked to glycerol by ether rather than an ester linkage. Some of these lipids have no phosphate groups, and therefore, they are not phospholipids. In other species, the cell membrane is made up of a lipid monolayer consisting of long lipids (about twice as long as a phospholipid) with glycerol ethers at both ends (diglycerol tetraethers). The molecules orient themselves with the polar glycerol groups on the surfaces and the nonpolar hydrocarbon chain in the interior. These unusual lipids contribute to the ability of many Archaea to grow under environmental conditions such as high salt, low pH, or very high temperature. B. Function The major functions of the cytoplasmic membrane are (1) selective permeability and transport of solutes; (2) electron transport and oxidative phosphorylation in aerobic species; (3) excretion of hydrolytic exoenzymes; (4) contain the enzymes and carrier molecules that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids; and (5) bear the receptors and other proteins of the chemotactic and other sensory transduction systems. 1. Permeability and transport The cytoplasmic membrane forms a hydrophobic barrier impermeable to most hydrophilic molecules. However, several mechanisms (transport systems) exist that enable the cell to transport nutrients into and waste products out of the cell. These transport systems work against a concentration gradient to increase the nutrient concentrations inside the cell, a function that requires energy in some form. There are three general transport mechanisms involved in membrane transport: passive transport, active transport, and group translocation. a. Passive transport—This mechanism relies on diffusion, uses no energy, and operates only when the solute is at higher concentration outside than inside the cell. Simple diffusion accounts for the entry of very few nutrients, including dissolved oxygen, carbon dioxide, and water itself. Simple diffusion provides neither speed nor selectivity. Facilitated diffusion also uses no energy, so the solute never achieves an internal concentration greater than what exists outside the cell. However, facilitated diffusion is selective. Channel proteins form selective channels that facilitate the passage of specific molecules. Facilitated diffusion is common in eukaryotic microorganisms (eg, yeast) but is rare in prokaryotes. Glycerol is one of the few compounds that enters prokaryotic cells by facilitated diffusion. b. Active transport—Many nutrients are concentrated more than a thousandfold as a result of active transport. There are two types of active transport mechanisms depending on the source of energy used: ion­coupled transport and ATP­binding cassette (ABC) transport. 1) Ion­coupled transport—These systems move a molecule across the cell membrane at the expense of a previously established ion gradient such as proton­ or sodium­motive force. There are three basic types: uniport, symport, and antiport (Figure 2­14). Ion­coupled transport is particularly common in aerobic organisms, which have an easier time generating an ion­motive force than do anaerobes. Uniporters catalyze the transport of a substrate independent of any coupled ion. Symporters catalyze the simultaneous transport of two substrates in the same direction by a single carrier; for example, an H+ gradient can permit symport of an oppositely charged ion (eg, glycine) or a neutral molecule (eg, galactose). Antiporters catalyze the simultaneous transport of two like­charged compounds in opposite directions by a common carrier (eg, H+:Na+). Approximately, 40% of the substrates transported by E. coli use this mechanism. Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 FIGURE Chapter2­14 2: Cell Structure, Page 15 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility Three types of porters: A : uniporters, B : symporters, and C : antiporters. Uniporters catalyze the transport of a single species independently of any other, symporters catalyze the cotransport of two dissimilar species (usually a solute and a positively charged ion, H+) in the same direction, and common in aerobic organisms, which have an easier time generating an ion­motive force than do anaerobes. Uniporters catalyze the transport of a substrate independent of any coupled ion. Symporters catalyze the simultaneous transport of two substrates in the same directionNaresuan by a singleUniversity carrier; for example, an H+ gradient can permit symport of an oppositely charged ion (eg, glycine) or a neutral molecule (eg, galactose). Antiporters catalyze Access Provided by: the simultaneous transport of two like­charged compounds in opposite directions by a common carrier (eg, H+:Na+). Approximately, 40% of the substrates transported by E. coli use this mechanism. FIGURE 2­14 Three types of porters: A : uniporters, B : symporters, and C : antiporters. Uniporters catalyze the transport of a single species independently of any other, symporters catalyze the cotransport of two dissimilar species (usually a solute and a positively charged ion, H+) in the same direction, and antiporters catalyze the exchange transport of two similar solutes in opposite directions. A single transport protein may catalyze just one of these processes, two of these processes, or even all three of these processes, depending on conditions. Uniporters, symporters, and antiporters have been found to be structurally similar and evolutionarily related, and they function by similar mechanisms. (Reproduced with permission from Saier MH Jr: Peter Mitchell and his chemiosmotic theories. ASM News 1997;63:13.) 2) ABC transport—This mechanism uses ATP directly to transport solutes into the cell. In Gram­negative bacteria, the transport of many nutrients is facilitated by specific binding proteins located in the periplasmic space; in Gram­positive cells, the binding proteins are attached to the outer surface of the cell membrane. These proteins function by transferring the bound substrate to a membrane­bound protein complex. Hydrolysis of ATP is then triggered, and the energy is used to open the membrane pore and allow the unidirectional movement of the substrate into the cell. Approximately 40% of the substrates transported by E. coli use this mechanism. c. Group translocation—In addition to true transport, in which a solute is moved across the membrane without change in structure, bacteria use a Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 process called Chapter 2: Cellgroup translocation (vectorial metabolism) to effect the net uptake of certain sugars (eg, glucose and mannose), the substrate Structure, Page 16 / 44 ©2024 McGraw becoming Hill. All Rights phosphorylated Reserved. during Terms the transport of UseIn aPrivacy process. Policygroup strict sense, Notice Accessibility translocation is not active transport because no concentration gradient is involved. This process allows bacteria to use their energy resources efficiently by coupling transport with metabolism. In this process, a membrane carrier protein is first phosphorylated in the cytoplasm at the expense of phosphoenolpyruvate; the phosphorylated carrier protein then facilitated by specific binding proteins located in the periplasmic space; in Gram­positive cells, the binding proteins are attached to the outer surface Naresuan University of the cell membrane. These proteins function by transferring the bound substrate to a membrane­bound protein complex. Hydrolysis of ATP is then Access Provided by: triggered, and the energy is used to open the membrane pore and allow the unidirectional movement of the substrate into the cell. Approximately 40% of the substrates transported by E. coli use this mechanism. c. Group translocation—In addition to true transport, in which a solute is moved across the membrane without change in structure, bacteria use a process called group translocation (vectorial metabolism) to effect the net uptake of certain sugars (eg, glucose and mannose), the substrate becoming phosphorylated during the transport process. In a strict sense, group translocation is not active transport because no concentration gradient is involved. This process allows bacteria to use their energy resources efficiently by coupling transport with metabolism. In this process, a membrane carrier protein is first phosphorylated in the cytoplasm at the expense of phosphoenolpyruvate; the phosphorylated carrier protein then binds the free sugar at the exterior membrane face and transports it into the cytoplasm, releasing it as a sugar phosphate. Such systems of sugar transport are called phosphotransferase systems. Phosphotransferase systems are also involved in movement toward these carbon sources (chemotaxis) and in the regulation of several other metabolic pathways (catabolite repression). d. Special transport processes—Iron (Fe) is an essential nutrient for the growth of almost all bacteria. Under anaerobic conditions, Fe is generally in the +2 oxidation state and soluble. However, under aerobic conditions, Fe is generally in the +3 oxidation state and insoluble. The internal compartments of animals contain virtually no free Fe; it is sequestered in complexes with such proteins as transferrin and lactoferrin. Some bacteria solve this problem by secreting siderophores—compounds that chelate Fe and promote its transport as a soluble complex. One major group of siderophores consists of derivatives of hydroxamic acid (−CONH2OH), which chelate Fe3+ very strongly. The iron–hydroxamate complex is actively transported into the cell by the cooperative action of a group of proteins that span the outer membrane, periplasm, and inner membrane. The iron is released, and the hydroxamate can exit the cell and be used again for iron transport. Some pathogenic bacteria use a fundamentally different mechanism involving specific receptors that bind host transferrin and lactoferrin (as well as other iron­containing host proteins). The Fe is removed and transported into the cell using an ABC transporter. 2. Electron transport and oxidative phosphorylation The cytochromes and other enzymes and components of the respiratory chain, including certain dehydrogenases, are located in the cytoplasmic membrane. The bacterial cytoplasmic membrane is thus a functional analog of the mitochondrial membrane—a relationship which has been taken by many biologists to support the theory that mitochondria have evolved from symbiotic bacteria. The mechanism by which ATP generation is coupled to electron transport is discussed in Chapter 6. 3. Excretion of hydrolytic exoenzymes and pathogenicity proteins All organisms that rely on macromolecular organic polymers as a source of nutrients (eg, proteins, polysaccharides, and lipids) excrete hydrolytic enzymes that degrade these polymers to subunits small enough to penetrate the cell membrane. Higher animals secrete such enzymes into the lumen of the digestive tract; bacteria (both Gram­positive and Gram­negative) secrete them directly into the external medium or into the periplasmic space between the peptidoglycan layer and the outer membrane of the cell wall in the case of Gram­negative bacteria (see The Cell Wall). In Gram­positive bacteria, proteins are secreted directly across the cytoplasmic membrane, but in Gram­negative bacteria, secreted proteins must traverse the outer membrane as well. At least six pathways of protein secretion have been described in bacteria: the type I, type II, type III, type IV, type V, and type VI secretion systems. A schematic overview of the type I to V systems is presented in Figure 2­15. The type I and IV secretion systems have been described in both Gram­negative and Gram­positive bacteria, but the type II, III, V, and VI secretion systems have been found only in Gram­ negative bacteria. Proteins secreted by the type I and III pathways traverse the inner (cytoplasmic) membrane (IM) and outer membrane (OM) in one step, but proteins secreted by the type II and V pathways cross the IM and OM in separate steps. Proteins secreted by the type II and V pathways are synthesized on cytoplasmic ribosomes as preproteins containing an extra leader or signal sequence of 15–40 amino acids—most commonly about 30 amino acids—at the amino terminal and require the sec system for transport across the IM. In E. coli, the sec pathway comprises a number of IM proteins (SecD to SecF, SecY), a cell membrane­associated ATPase (SecA) that provides energy for export, a chaperone (SecB) that binds to the preprotein, and the periplasmic signal peptidase. After translocation, the leader sequence is cleaved off by the membrane­bound signal peptidase, and the mature protein is released into the periplasmic space. In contrast, proteins secreted by the type I and III systems do not have a leader sequence and are exported intact. FIGURE 2­15 The protein secretion systems of Gram­negative bacteria. Five secretion systems of Gram­negative bacteria are shown. The Sec­dependent and Tat pathways deliver proteins from the cytoplasm to the periplasmic space. The type II, type V, and sometimes type IV systems complete the secretion process begun by the Sec­dependent pathway. The Tat system appears to deliver proteins only to the type II pathway. The type I and III systems bypass Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 the Sec­dependent Chapter and Tat pathways, moving proteins directly from the cytoplasm, through the outer membrane, to the extracellular space. 2: Cell Structure, The Page 17type / 44 IV system ©2024 can work McGraw Hill.either with the All Rights Sec­dependent Reserved. Termspathway of Use or can work Privacy alone Policy to transport Notice proteins to the extracellular space. Proteins translocated by Accessibility the Sec­dependent pathway and the type III pathway are delivered to those systems by chaperone proteins. ADP, adenosine diphosphate; ATP, adenosine triphosphate; EFGY; PuIS; SecD; TolC; Yop. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, FIGURE 2­15 Naresuan University Access Provided by: The protein secretion systems of Gram­negative bacteria. Five secretion systems of Gram­negative bacteria are shown. The Sec­dependent and Tat pathways deliver proteins from the cytoplasm to the periplasmic space. The type II, type V, and sometimes type IV systems complete the secretion process begun by the Sec­dependent pathway. The Tat system appears to deliver proteins only to the type II pathway. The type I and III systems bypass the Sec­dependent and Tat pathways, moving proteins directly from the cytoplasm, through the outer membrane, to the extracellular space. The type IV system can work either with the Sec­dependent pathway or can work alone to transport proteins to the extracellular space. Proteins translocated by the Sec­dependent pathway and the type III pathway are delivered to those systems by chaperone proteins. ADP, adenosine diphosphate; ATP, adenosine triphosphate; EFGY; PuIS; SecD; TolC; Yop. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw­Hill; 2008. © McGraw­Hill Education.) In Gram­negative and Gram­positive bacteria, another cytoplasmic membrane system that uses the twin­arginine targeting translocase (tat pathway) can move proteins across the IM. In Gram­negative bacteria, these proteins are then delivered to the type II system (Figure 2­15). The tat pathway is distinct from the sec system in that it translocates already folded proteins. Although proteins secreted by the type II and V systems are similar in the mechanism by which they cross the IM, differences exist in how they traverse the OM. Proteins secreted by the type II system are transported across the OM by a multiprotein complex (see Figure 2­15). This is the primary pathway for the secretion of extracellular degradative enzymes by Gram­negative bacteria. Elastase, phospholipase C, and exotoxin A are secreted by this system in Pseudomonas aeruginosa. However, proteins secreted by the type V system autotransport across the outer membrane by virtue of a carboxyl terminal sequence, which is enzymatically removed upon release of the protein from the OM. Some extracellular proteins—eg, the IgA protease of Neisseria gonorrhoeae and the vacuolating cytotoxin of Helicobacter pylori—are secreted by this system. The type I and III secretion pathways are sec independent and thus do not involve amino terminal processing of the secreted proteins. Protein secretion by these pathways occurs in a continuous process without the presence of a cytoplasmic intermediate. Type I secretion is exemplified by the α­hemolysin of E. coli and the adenylyl cyclase of Bordetella pertussis. Type I secretion requires three secretory proteins: an IM ATP­binding cassette (ABC transporter), which provides energy for protein secretion; an OM protein; and a membrane fusion protein, which is anchored in the inner membrane and spans the periplasmic space (see Figure 2­15). Instead of a signal peptide, the information is located within the carboxyl terminal 60 amino acids of the secreted protein. The type III secretion pathway is a contact­dependent system. It is activated by contact with a host cell, and then injects a toxin protein into the host cell directly. The type III secretion apparatus is composed of approximately 20 proteins, most of which are located in the IM. Many of these IM components are homologous to the flagellar biosynthesis apparatus of both Gram­negative and Gram­positive bacteria. As in type I secretion, the proteins secreted via the type III pathway are not subject to amino terminal processing during secretion. Type IV pathways secrete either polypeptide toxins (directed against eukaryotic cells) or protein–DNA complexes either between two bacterial cells or between a bacterial and a eukaryotic cell. Type IV secretion is exemplified by the protein–DNA complex delivered by Agrobacterium tumefaciens into a plant cell. Additionally, B. pertussis and H. pylori possess type IV secretion systems that mediate secretion of pertussis toxin and interleukin­8–inducing factor, respectively. The sec­independent type VI secretion was recently described in P. aeruginosa, where it contributes to pathogenicity in patients with cystic fibrosis. Downloaded This12:7 2024­8­4 secretion system A Your IP isis110.170.245.47 composed of 15–20 proteins whose biochemical functions are not well understood. However, recent studies suggest Chapter 2: Cell that some of these proteins share homology with bacteriophage tail proteins. Structure, Page 18 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility The characteristics of the protein secretion systems of bacteria are summarized in Table 9­5.  Naresuan Type IV pathways secrete either polypeptide toxins (directed against eukaryotic cells) or protein–DNA complexes either between two bacterialUniversity cells or between a bacterial and a eukaryotic cell. Type IV secretion is exemplified by the protein–DNA complex delivered by AgrobacteriumAccess Provided by: tumefaciens into a plant cell. Additionally, B. pertussis and H. pylori possess type IV secretion systems that mediate secretion of pertussis toxin and interleukin­8–inducing factor, respectively. The sec­independent type VI secretion was recently described in P. aeruginosa, where it contributes to pathogenicity in patients with cystic fibrosis. This secretion system is composed of 15–20 proteins whose biochemical functions are not well understood. However, recent studies suggest that some of these proteins share homology with bacteriophage tail proteins. The characteristics of the protein secretion systems of bacteria are summarized in Table 9­5. 4. Biosynthetic functions The cell membrane is the site of the carrier lipids on which the subunits of the cell wall are assembled (see the discussion of synthesis of cell wall substances in Chapter 6) as well as of the enzymes of cell wall biosynthesis. The enzymes of phospholipid synthesis are also localized in the cell membrane. 5. Chemotactic systems Attractants and repellents bind to specific receptors in the bacterial membrane (see Flagella). There are at least 20 different chemoreceptors in the membrane of E. coli, some of which also function as a first step in the transport process. The Cell Wall The internal osmotic pressure of most bacteria ranges from 5 to 20 atm as a result of solute concentration via active transport. In most environments, this pressure would be sufficient to burst the cell were it not for the presence of a high­tensile­strength cell wall (Figure 2­16). The bacterial cell wall owes its strength to a layer composed of a substance variously referred to as murein, mucopeptide, or peptidoglycan (all, including “cell wall,” are synonyms). The structure of peptidoglycan is discussed as follows. FIGURE 2­16 The rigid cell wall determines the shape of the bacterium. Even though the cell has split apart, the cell wall maintains its original shape. (Courtesy of Dale C. Birdsell.) Most bacteria are classified as Gram­positive or Gram­negative according to their response to the Gram­staining procedure. This procedure was named for the histologist Hans Christian Gram, who developed this differential staining procedure in an attempt to identify bacteria in infected tissues. The Gram­stain depends on the ability of certain bacteria (the Gram­positive bacteria) to retain a complex of crystal violet (a purple dye) and iodine after a brief wash with alcohol or acetone. Gram­negative bacteria do not retain the dye–iodine complex and become translucent, but they can then be counterstained with safranin (a red dye). Thus, Gram­positive bacteria look purple under the microscope, and Gram­negative bacteria look red. The distinction between these two groups turns out to reflect fundamental differences in their cell envelopes (Table 2­1). TABLE 2­1 Comparison of Features of Gram­Positive and Gram­Negative Bacteria Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 19 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility The Gram­stain depends on the ability of certain bacteria (the Gram­positive bacteria) to retain a complex of crystal violet (a purple dye) and iodine Naresuan University after a brief wash with alcohol or acetone. Gram­negative bacteria do not retain the dye–iodine complex and become translucent, but they can then be Access Provided by: counterstained with safranin (a red dye). Thus, Gram­positive bacteria look purple under the microscope, and Gram­negative bacteria look red. The distinction between these two groups turns out to reflect fundamental differences in their cell envelopes (Table 2­1). TABLE 2­1 Comparison of Features of Gram­Positive and Gram­Negative Bacteria Gram­Positive Gram­Negative Color of Gram­Stained Cell Purple Reddish­pink Representative Genera Bacillus, Staphylococcus, Streptococcus Escherichia, Neisseria, Pseudomonas Distinguishing Structures/Components Peptidoglycan Thick layer Thin layer Teichoic acids Present Absent Outer membrane Absent Present Lipopolysaccharide Absent Present (endotoxin) Porin proteins Absent (unnecessary because there is no outer Present; allow passage of molecules through outer membrane) membrane Periplasm Absent Present General Characteristics Sensitivity to penicillin Generally more susceptible (with notable exceptions) Generally less susceptible (with notable exceptions) Sensitivity to lysozyme Yes No In addition to providing osmotic protection, the cell wall plays an essential role in cell division as well as serving as a primer for its own biosynthesis. The cell wall is, in general, nonselectively permeable; one layer of the Gram­negative wall, however—the outer membrane—hinders the passage of relatively large molecules (see next). The biosynthesis of the cell wall and the antibiotics that interfere with this process are discussed in Chapter 6. A. The Peptidoglycan Layer Peptidoglycan is a complex polymer consisting, for the purposes of description, of three parts: a backbone, composed of alternating N­ acetylglucosamine and N­acetylmuramic acid connected by β1→4 linkages; a set of identical tetrapeptide side chains attached to N­acetylmuramic acid; and a set of identical peptide cross­bridges (Figure 2­17). The backbone is the same in all bacterial species; the tetrapeptide side chains and the peptide cross­bridges vary from species to species. In many Gram­negative cell walls, the cross­bridge consists of a direct peptide linkage between the diaminopimelic acid (DAP) amino group of one side chain and the carboxyl group of the terminal D­alanine of a second side chain. FIGURE 2­17 Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 20 / 44 ©2024 McGraw Components andHill. All Rights structure Reserved. Terms of peptidoglycan. of Use structure A : Chemical Privacy of Policy Notice Accessibility N­acetylglucosamine (NAG) and N­acetylmuramic acid (NAM); the ring structures of the two molecules are glucose. Glycan chains are composed of alternating subunits of NAG and NAM joined by covalent bonds. Adjacent glycan chains are cross­linked via their tetrapeptide chains to create peptidoglycan. B : Interconnected glycan chains form a very large three­dimensional acetylglucosamine and N­acetylmuramic acid connected by β1→4 linkages; a set of identical tetrapeptide side chains attached to N­acetylmuramic Naresuan University acid; and a set of identical peptide cross­bridges (Figure 2­17). The backbone is the same in all bacterial species; the tetrapeptide side chains and the Access Provided by: peptide cross­bridges vary from species to species. In many Gram­negative cell walls, the cross­bridge consists of a direct peptide linkage between the diaminopimelic acid (DAP) amino group of one side chain and the carboxyl group of the terminal D­alanine of a second side chain. FIGURE 2­17 Components and structure of peptidoglycan. A : Chemical structure of N­acetylglucosamine (NAG) and N­acetylmuramic acid (NAM); the ring structures of the two molecules are glucose. Glycan chains are composed of alternating subunits of NAG and NAM joined by covalent bonds. Adjacent glycan chains are cross­linked via their tetrapeptide chains to create peptidoglycan. B : Interconnected glycan chains form a very large three­dimensional molecule of peptidoglycan. The β1→4 linkages in the backbone are cleaved by lysozyme. (Reproduced with permission from Nester EW, Anderson DG, Roberts CE, et al: Microbiology: A Human Perspective, 6th ed. McGraw­Hill, 2009. © McGraw­Hill Education.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 21 / 44 ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility  Naresuan University Access Provided by: The tetrapeptide side chains of all species, however, have certain notable features in common. Most have L­alanine at position 1 (attached to N­ acetylmuramic acid), D­glutamate or substituted D­glutamate at position 2, and D­alanine at position 4. Position 3 is the most variable one: Most Gram­ negative bacteria have diaminopimelic acid at this position, to which is linked the lipoprotein cell wall component discussed as follows. Gram­positive bacteria usually have L­lysine at position 3; however, some may have diaminopimelic acid or another amino acid at this position. Diaminopimelic acid is a unique element of bacterial cell walls. It is never found in the cell walls of Archaea or eukaryotes. Diaminopimelic acid is the immediate precursor of lysine in the bacterial biosynthesis of that amino acid (see Figure 6­19). Bacterial mutants that are blocked before diaminopimelic acid in the biosynthetic pathway grow normally when provided with diaminopimelic acid in the medium; when given L­lysine alone, however, they lyse, because they continue to grow but are specifically unable to make new cell wall peptidoglycan. The fact that all peptidoglycan chains are cross­linked means that each peptidoglycan layer is a single giant molecule. In Gram­positive bacteria, there are as many as 40 sheets of peptidoglycan, comprising up to 50% of the cell wall material; in Gram­negative bacteria, there appears to be only one or two sheets, comprising 5–10% of the wall material. Bacteria owe their shapes, which are characteristic of particular species, to their cell wall structure. B. Special Components of Gram­Positive Cell Walls Most Gram­positive cell walls contain considerable amounts of teichoic and teichuronic acids, which may account for up to 50% of the dry weight of the wall and 10% of the dry weight of the total cell. In addition, some Gram­positive walls may contain polysaccharide molecules. 1. Teichoic and teichuronic acids The term teichoic acids encompass all wall, membrane, or capsular polymers containing glycerophosphate or ribitol phosphate residues. These polyalcohols are connected by phosphodiester linkages and usually have other sugars and D­alanine attached (Figure 2­18A). Because they are negatively charged, teichoic acids are partially responsible for the net negative charge of the cell surface. There are two types of teichoic acids: wall teichoic acid (WTA), covalently linked to peptidoglycan; and membrane teichoic acid, covalently linked to membrane glycolipid. Because the latter are intimately associated with lipids, they have been called lipoteichoic acids (LTA). Together with peptidoglycan, WTA and LTA make up a polyanionic network or matrix that provides functions relating to the elasticity, porosity, tensile strength, and electrostatic properties of the envelope. Although not all Gram­positive bacteria have conventional LTA and WTA, those that lack these polymers generally have functionally similar ones. FIGURE 2­18 A : Teichoic acid Downloaded structure. 2024­8­4 12:7The A segment Your IP of a teichoic acid made of phosphate, glycerol, and a side chain, R. R may represent D­alanine, glucose, or other is 110.170.245.47 molecules. B : Teichoic and lipoteichoic acids of the Gram­positive envelope. (Reproduced with permission from Willey JM, Sherwood LM,Page Chapter 2: Cell Structure, 22 / 44 Woolverton ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw­Hill; 2008. © McGraw­Hill Education.) latter are intimately associated with lipids, they have been called lipoteichoic acids (LTA). Together with peptidoglycan, WTA and LTA make up a polyanionic network or matrix that provides functions relating to the elasticity, porosity, tensile strength, and electrostatic properties of the envelope. Naresuan University Although not all Gram­positive bacteria have conventional LTA and WTA, those that lack these polymers generally have functionallyAccess similar ones. Provided by: FIGURE 2­18 A : Teichoic acid structure. The segment of a teichoic acid made of phosphate, glycerol, and a side chain, R. R may represent D­alanine, glucose, or other molecules. B : Teichoic and lipoteichoic acids of the Gram­positive envelope. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw­Hill; 2008. © McGraw­Hill Education.) Most teichoic acids contain substantial amounts of D­alanine, usually attached to position 2 or 3 of glycerol or position 3 or 4 of ribitol. In some of the more complex teichoic acids, however, D­alanine is attached to one of the sugar residues. In addition to D­alanine, other substituents may be attached to the free hydroxyl groups of glycerol and ribitol (eg, glucose, galactose, N­acetylglucosamine, N­acetylgalactosamine, or succinate). A given species may have more Downloaded than one12:7 2024­8­4 typeAofYour sugarIPsubstituent in addition to D­alanine; in such cases, it is not certain whether the different sugars occur on the is 110.170.245.47 Chapter 2: Cell Structure, Page 23 / 44 same or on separate teichoic acid molecules. The composition of the teichoic acid formed by a given bacterial species can vary with the composition of ©2024 McGraw Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility the growth medium. The teichoic acids constitute major surface antigens of those Gram­positive species that possess them, and their accessibility to antibodies has been  Naresuan University Access Provided by: Most teichoic acids contain substantial amounts of D­alanine, usually attached to position 2 or 3 of glycerol or position 3 or 4 of ribitol. In some of the more complex teichoic acids, however, D­alanine is attached to one of the sugar residues. In addition to D­alanine, other substituents may be attached to the free hydroxyl groups of glycerol and ribitol (eg, glucose, galactose, N­acetylglucosamine, N­acetylgalactosamine, or succinate). A given species may have more than one type of sugar substituent in addition to D­alanine; in such cases, it is not certain whether the different sugars occur on the same or on separate teichoic acid molecules. The composition of the teichoic acid formed by a given bacterial species can vary with the composition of the growth medium. The teichoic acids constitute major surface antigens of those Gram­positive species that possess them, and their accessibility to antibodies has been taken as evidence that they lie on the outside surface of the peptidoglycan. Their activity is often increased, however, by partial digestion of the peptidoglycan; thus, much of the teichoic acid may lie between the cytoplasmic membrane and the peptidoglycan layer, possibly extending upward through pores in the latter (Figure 2­18B). In the pneumococcus (Streptococcus pneumoniae), the teichoic acids bear the antigenic determinants called Forssman antigen. In Streptococcus pyogenes, LTA is associated with the M protein that protrudes from the cell membrane through the peptidoglycan layer. The long M protein molecules together with the LTA form microfibrils that facilitate the attachment of S. pyogenes to animal cells (see Chapter 14). The teichuronic acids are similar polymers, but the repeat units include sugar acids (eg, N­acetylmannosuronic or D­glucosuronic acid) instead of phosphoric acids. They are synthesized in place of teichoic acids when phosphate is limiting. 2. Polysaccharides The hydrolysis of Gram­positive walls has yielded, from certain species, neutral sugars, such as mannose, arabinose, rhamnose, and glucosamine, and acidic sugars, such as glucuronic acid and mannuronic acid. It has been proposed that these sugars exist as subunits of polysaccharides in the cell wall; the discovery, however, that teichoic and teichuronic acids may contain a variety of sugars (see Figure 2­18A) leaves the true origin of these sugars uncertain. C. Special Components of Gram­Negative Cell Walls Gram­negative cell walls contain three components that lie outside of the peptidoglycan layer: outer membrane, lipopolysaccharide, and lipoprotein (Figure 2­19). FIGURE 2­19 Molecular representation of the envelope of a Gram­negative bacterium. Ovals and rectangles represent sugar residues, and circles depict the polar head groups of the glycerophospholipids (phosphatidylethanolamine and phosphatidylglycerol). The core region shown is that of E. coli K­12, a strain that does not normally contain an O­antigen repeat unless transformed with an appropriate plasmid. MDO, membrane­derived oligosaccharides. (Reproduced with permission from Raetz CRH: Bacterial endotoxins: Extraordinary lipids that activate eucaryotic signal transduction. J Bacteriol 1993;175:5745.) Downloaded 2024­8­4 12:7 A Your IP is 110.170.245.47 Chapter 2: Cell Structure, Page 24 / 44 1. OuterMcGraw ©2024 membrane Hill. All Rights Reserved. Terms of Use Privacy Policy Notice Accessibility The outer membrane is chemically distinct from all other biological membranes. It is a bilayered structure; its inner leaflet resembles in composition head groups of the glycerophospholipids (phosphatidylethanolamine and phosphatidylglycerol). The core region shown is that of E. coli K­12, a strain Naresuan University that does not normally contain an O­antigen repeat unless transformed with an appropriate plasmid. MDO, membrane­derived oligosaccharides. Access Provided by: (Reproduced with permission from Raetz CRH: Bacterial endotoxins: Extraordinary lipids that activate eucaryotic signal transduction. J Bacteriol 1993;175:5745.) 1. Outer membrane The outer membrane is chemically distinct from all other biological membranes. It is a bilayered structure; its inner leaflet resembles in composition that of the cytoplasmic membrane, and its outer leaflet contains a distinctive component, a lipopolysaccharide (LPS) (see next). As a result, this is an asymmetrical membrane, and the properties of this bilayer differ considerably from those of a symmetrical biologic membrane such as the cell membrane. The ability of the outer membrane to exclude hydrophobic molecules is an unusual feature among biologic membranes and serves to protect the cell (in the case of enteric bacteria) from deleterious substances such as bile salts. Because of its lipid nature, the outer membrane would be expected to exclude hydrophilic molecules as well. However, the outer membrane has special channels, consisting of protein molecules called porins that permit the passive diffusion of low­molecular­weight hydrophilic compounds, such as sugars, amino acids, and certain ions. Large antibiotic molecules penetrate the outer membrane relatively slowly, which accounts for the relatively high resistance of Gram­negative bacteria to some antibiotics. The permeability of the outer membrane varies widely from one Gram­negative species to another; in P. aeruginosa, for example, which is extremely resistant to antibacterial agents, the outer membrane is 100 times less permeable than that of E. coli. The major proteins of the outer membrane, named according to the genes that code for them, have been placed into several functional categories on the basis of mutants in which they are lacking and on the basis of experiments in which purified proteins have been reconstituted into artificial membranes. Porins, exemplified by OmpC, D, and F and PhoE of E. coli and Salmonella typhimurium, are trimeric proteins that penetrate both the i