Microbial Cell Structure and Function Review 1.pdf

CHAPTER 2 Microbial Cell Structure and Function microbiologynow Archaeal Tortoise and Hare Motility is important for microorganisms because the ability to move allows cells to explore new habitats and exploit their resources. Motility has been studied for over 50 years in the flag...

CHAPTER 2 Microbial Cell Structure and Function microbiologynow Archaeal Tortoise and Hare Motility is important for microorganisms because the ability to move allows cells to explore new habitats and exploit their resources. Motility has been studied for over 50 years in the flagellated bacterium Escherichia coli. It is with E. coli that scientists first discovered that the bacterial flagellum functions by rotating and that when speed is expressed in terms of body lengths traveled per second, swimming E. coli cells are actually moving faster than the fastest animals. Studies of the archaeon Halobacterium showed that its flagella also rotate but that they were thinner than their bacterial counterparts and were composed of a protein distinct from flagellin, the protein that makes up bacterial flagella. Moreover, observations of swimming cells showed that Halobacterium was a slowpoke, moving at less than one-tenth the speed of E. coli. This raised the interesting question of whether this was true of all Archaea; are they naturally joggers instead of sprinters? Microbiologists recently zeroed in on the movements of I Microscopy 26 swimming Archaea and showed that Halobacterium was the II Cells of Bacteria and Archaea 32 slowest of all species examined.1 By contrast, cells of the archaeon Methanocaldococcus (cells with flagellar tufts in III The Cytoplasmic Membrane and photo) swam nearly 50 times faster than cells of Halobacterium Transport 35 and 10 times faster than cells of E. coli. Astonishingly, IV Cell Walls of Bacteria and Archaea 41 Methanocaldococcus moves at nearly 500 cell lengths per V Other Cell Surface Structures and second, which makes it the fastest organism on Earth! The thin diameter of the archaeal flagellum obviously does Inclusions 48 not mandate a slow swimming speed as some had predicted VI Microbial Locomotion 56 from the Halobacterium work. Instead, swimming speeds of VII Eukaryotic Microbial Cells 64 Archaea can and do vary greatly.1 Indeed, the existence of both a “tortoise” and a “hare” within the Archaea shows that there is still much to learn about the structure and function of microbial cells. 1 Herzog, B., and R. Wirth. 2012. Swimming behavior of selected species of Archaea. Appl. Environ. Microbiol. 78: 1670–1674. 25 26 U N I T 1 T H E F O U N D AT I O N S O F M I C R O B I O L O G Y I Microscopy H istorically, the science of microbiology has taken its greatest leaps forward as new tools for the study of microorganisms are developed and old tools improve. The microscope is the microbi- We begin with the light microscope, for which the limits of res- olution are about 0.2 mm (mm is the abbreviation for micrometer, 10-6 m). We then proceed to the electron microscope, for which ologist’s oldest and most basic tool for studying microbial struc- resolution is considerably greater. ture. Many types of microscopy are used and some are extremely powerful. So as a prelude to our study of cell structure, let’s first The Compound Light Microscope take a look at some common tools for visualizing cells with a goal The light microscope uses visible light to illuminate cell struc- of understanding how they work and what they can tell us. tures. Several types of light microscopes are used in microbiol- ogy: bright-field, phase-contrast, differential interference contrast, 2.1 Discovering Cell Structure: dark-field, and fluorescence. With the bright-field microscope, specimens are visualized be- Light Microscopy cause of the slight differences in contrast that exist between them To see microorganisms, one needs a microscope of some sort, and their surroundings, differences that arise because cells absorb either a light microscope or an electron microscope. In general, or scatter light to varying degrees. The modern compound light light microscopes are used to examine cells at relatively low mag- microscope contains two lenses, objective and ocular, that func- nifications, and electron microscopes are used to examine cells tion in combination to form the image. The light source is focused and cell structures at very high magnification. on the specimen by the condenser (Figure 2.1). Bacterial cells are All microscopes employ lenses that magnify the image. Mag- typically difficult to see well with the bright-field microscope be- nification, however, is not the limiting factor in our ability to see cause the cells themselves lack significant contrast with their sur- small objects. It is instead resolution—the ability to distinguish rounding medium. Cells visualized by a form of light microscopy two adjacent objects as distinct and separate—that governs our called phase-contrast (Section 2.2; see inset Figure 2.1) overcome ability to see the very small. Although magnification can be in- these limitations. Pigmented microorganisms are also an excep- creased virtually without limit, resolution cannot, because reso- tion because the color of the organism itself adds contrast, which lution is a function of the physical properties of light. makes them easier to visualize by bright-field optics (Figure 2.2). Magnification Light path Marie Asao and 100×, 400×, M.T. Madigan Visualized 1000× image Eye Ocular lenses Specimen on glass slide 10× Ocular lens Intermediate image (inverted from that Objective lens of the specimen) Stage Condenser 10×, 40× or Objective lens 100×(oil) Focusing knobs Specimen None Condenser lens Light source Carl Zeiss, Inc. Light source (a) (b) Figure 2.1 Microscopy. (a) A compound light microscope (inset photomicrograph of unstained cells taken through a phase-contrast light microscope). (b) Path of light through a compound light microscope. Besides 10 * , ocular lenses are available in 15–30 * magnifications. Figure 2.5 compares cells visualized by bright field with those by phase contrast.  CHAPTER 2 MICROBIAL CELL STRUCTURE AND FUNCTION 27 by allowing some of the light rays emerging from the specimen at angles (that would otherwise be lost to the objective lens) to be collected and viewed. UNIT 1 MINIQUIZ Define the terms magnification and resolution. T. D. Brock What is the upper limit of magnification for a bright-field microscope? Why is this so? (a) 2.2 Improving Contrast in Light Microscopy In light microscopy, improving contrast improves the final image. Staining is a quick and easy way to improve contrast, but there are many other ways to do this. Norbert Pfennig Staining: Increasing Contrast for Bright-Field Microscopy (b) Dyes can be used to stain cells and increase their contrast so that they can be more easily seen in the bright-field microscope. Dyes Figure 2.2 Bright-field photomicrographs of pigmented microorganisms. are organic compounds, and each class of dye has an affinity for (a) A green alga (eukaryote). The green structures are chloroplasts. (b) Purple specific cellular materials. Many dyes used in microbiology are phototrophic bacteria (prokaryote). The algal cell is about 15 mm wide, and the bacterial cells are about 5 mm wide. positively charged, and for this reason they are called basic dyes. Examples of basic dyes include methylene blue, crystal violet, and safranin. Basic dyes bind strongly to negatively charged cell com- For cells lacking pigments there are several ways to boost con- ponents, such as nucleic acids and acidic polysaccharides. Be- trast, and we consider these methods in the next section. cause cell surfaces tend to be negatively charged, these dyes also combine with high affinity to the surfaces of cells, and hence are Magnification and Resolution very useful general-purpose stains. The total magnification of a compound light microscope is the To perform a simple stain one begins with dried preparations product of the magnification of its objective and ocular lenses of cells (Figure 2.3). A clean glass slide containing a dried suspen- (Figure 2.1b). Magnifications of about 2000* are the upper limit sion of cells is flooded for a minute or two with a dilute solu- for light microscopes, and at magnifications above this, resolu- tion of a basic dye, rinsed several times in water, and blotted dry. tion does not improve. Resolution is a function of the wavelength Because their cells are so small, it is common to observe dried, of light used and a characteristic of the objective lens known as stained preparations of Bacteria or Archaea with a high-power its numerical aperture, a measure of light-gathering ability. There (oil-immersion) lens. is a correlation between the magnification of a lens and its nu- merical aperture; lenses with higher magnification typically have Differential Stains: The Gram Stain higher numerical apertures. The diameter of the smallest object Stains that render different kinds of cells different colors are resolvable by any lens is equal to 0.5λ/numerical aperture, where called differential stains. An important differential-staining pro- λ is the wavelength of light used. This formula reveals that resolu- cedure used in microbiology is the Gram stain (Figure 2.4). On the tion is highest when blue light is used to illuminate a specimen basis of their reaction in the Gram stain, Bacteria can be divided (blue light has shorter wavelengths than white or red light) and into two major groups: gram-positive and gram-negative. After the objective has a very high numerical aperture. Gram staining, gram-positive bacteria appear purple-violet and As mentioned, the highest resolution possible in a compound gram-negative bacteria appear pink (Figure 2.4b). The color dif- light microscope is about 0.2 mm. What this means is that two ference in the Gram stain arises because of differences in the cell objects that are closer together than 0.2 mm cannot be resolved wall structure of gram-positive and gram-negative cells, a topic as distinct and separate. Microscopes used in microbiology we will consider later. After staining with a basic dye such as crystal have ocular lenses that magnify 10–20 * and objective lenses violet that renders cells purple in color, treatment with ethanol that magnify 10–100 * (Figure 2.1b). At 1000* , objects 0.2 mm decolorizes gram-negative cells but not gram-positive cells. in diameter can just be resolved. With the 100* objective, and Following counterstaining with a different-colored stain, typically with certain other objectives of very high numerical aperture, the red stain safranin, the two cell types can be distinguished an optical grade oil is placed between the microscope slide and microscopically by their different colors (Figure 2.4b). the objective. Lenses on which oil is used are called oil-immersion The Gram stain is the most common staining procedure used in lenses. Immersion oil increases the light-gathering ability of a lens microbiology, and it is often done to begin the characterization of a 28 U N I T 1 T H E F O U N D AT I O N S O F M I C R O B I O L O G Y Procedure Result 1. Preparing a smear 1. Flood the heat-fixed smear with crystal violet for 1 min All cells purple Spread culture in thin Dry in air film over slide 2. Add iodine solution for 1 min 2. Heat fixing and staining All cells remain purple 3. Decolorize with alcohol briefly — about 20 sec Pass slide through Flood slide with stain; flame to heat fix rinse and dry Gram-positive cells are purple; gram-negative cells are colorless 3. Microscopy 4. Counterstain with G– safranin for 1 –2 min G+ 100× Gram-positive (G+) cells Slide Oil are purple; gram-negative (G–) cells are pink to red Place drop of oil on slide; examine with 100× objective (a) lens Figure 2.3 Staining cells for microscopic observation. Stains improve the contrast between cells and their background. Center: Same cells as shown in Molecular Probes, Inc., Eugene, Oregon Figure 2.1 inset but stained with a basic dye. newly isolated bacterium. If a fluorescent microscope is available, the Gram stain can be reduced to a one-step procedure; gram- Leon J. Lebeau positive and gram-negative cells fluoresce different colors when treated with a special chemical (Figure 2.4c). Phase-Contrast and Dark-Field Microscopy (b) (c) Although staining is widely used in light microscopy, staining Figure 2.4 The Gram stain. (a) Steps in the procedure. (b) Microscopic kills cells and can distort their features. Two forms of light mi- observation of gram-positive (purple) and gram-negative (pink) bacteria. The croscopy improve image contrast of unstained (and thus live) organisms are Staphylococcus aureus and Escherichia coli, respectively. (c) Cells cells. These are phase-contrast microscopy and dark-field mi- of Pseudomonas aeruginosa (gram-negative, green) and Bacillus cereus (gram- croscopy (Figure 2.5). The phase-contrast microscope in particu- positive, orange) stained with a one-step fluorescent staining method. This method lar is widely used in teaching and research for the observation of allows for differentiating gram-positive from gram-negative cells in a single living preparations. staining step. Phase-contrast microscopy is based on the principle that cells dif- fer in refractive index (a factor by which light is slowed as it passes background (Figure 2.5c). Resolution by dark-field microscopy is through a material) from their surroundings. Light passing through often better than by light microscopy, and some objects can be a cell thus differs in phase from light passing through the surround- resolved by dark-field that cannot be resolved by bright-field or ing liquid. This subtle difference is amplified by a device in the objec- even by phase-contrast microscopes. Dark-field microscopy is a tive lens of the phase-contrast microscope called the phase ring, particularly good way to observe microbial motility, as bundles resulting in a dark image on a light background (Figure 2.5b; see also of flagella (the structures responsible for swimming motility) are inset to Figure 2.1). The ring consists of a phase plate that amplifies often resolvable with this technique (see Figure 2.50a). the variation in phase to produce the higher-contrast image. In the dark-field microscope, light reaches the specimen from Fluorescence Microscopy the sides only. The only light that reaches the lens is that scattered The fluorescence microscope is used to visualize specimens by the specimen, and thus the specimen appears light on a dark that fluoresce, emitting light of one color after absorbing light  CHAPTER 2 MICROBIAL CELL STRUCTURE AND FUNCTION 29 UNIT 1 M.T. Madigan M.T. Madigan M.T. Madigan (a) (b) (c) Figure 2.5 Cells visualized by different types of light microscopy. The same field of cells of the yeast Saccharomyces cerevisiae visualized by (a) bright-field microscopy, (b) phase-contrast microscopy, and (c) dark-field microscopy. Cells average 8–10 mm wide. of another color (Figure 2.6). Cells fluoresce because they either electron microscope offers one solution to this problem, but contain naturally fluorescent substances such as chlorophyll or certain forms of light microscopy can also improve the three- other fluorescing components (autofluorescence, Figure 2.6a, b), dimensional perspective of an image. or because they have been stained with a fluorescent dye (Figure 2.6c). DAPI (4′,6-diamidino-2-phenylindole) is a widely used flu- Differential Interference Contrast Microscopy orescent dye. DAPI stains cells bright blue because it complexes Differential interference contrast (DIC) microscopy is a form of with the cell’s DNA (Figure 2.6c). DAPI can be used to visualize light microscopy that employs a polarizer in the condenser to pro- cells in their natural habitats, such as soil, water, food, or a clini- duce polarized light (light in a single plane). The polarized light cal specimen. Fluorescence microscopy using DAPI is therefore then passes through a prism that generates two distinct beams. widely used in clinical diagnostic microbiology and also in micro- These beams pass through the specimen and enter the objective bial ecology for enumerating bacteria in a natural environment or lens where they are recombined into one. Because the two beams in a cell suspension. pass through substances that differ in refractive index, the com- bined beams are not totally in phase but instead interfere with MINIQUIZ each other, and this effect enhances subtle differences in cell structure. Thus, by DIC microscopy, cellular structures such as What color will a gram-negative cell be after Gram staining by the conventional method? the nucleus of eukaryotic cells (Figure 2.7 or endospores, vacuoles, and inclusions of bacterial cells, appear more three-dimensional. What major advantage does phase-contrast microscopy have DIC microscopy is typically used on unstained cells as it can over staining? reveal internal cell structures that are nearly invisible by bright- How can cells be made to fluoresce? field without the need for staining (compare Figure 2.5a with Figure 2.7). 2.3 Imaging Cells in Three Dimensions Confocal Scanning Laser Microscopy Thus far we have considered forms of microscopy in which the A confocal scanning laser microscope (CSLM) is a computer- rendered images are two-dimensional. How can this limitation controlled microscope that couples a laser to a fluorescent be overcome? We will see in the next section that the scanning microscope. The laser generates a bright three-dimensional R. W. Castenholz R. W. Castenholz Nancy J. Trun (a) (b) (c) Figure 2.6 Fluorescence microscopy. (a, b) Cyanobacteria. The same cells are observed by bright-field microscopy in part a and by fluorescence microscopy in part b. The cells fluoresce red because they contain chlorophyll a and other pigments. (c) Fluorescence photomicrograph of cells of Escherichia coli made fluorescent by staining with the fluorescent dye DAPI, which binds to DNA. 30 U N I T 1 T H E F O U N D AT I O N S O F M I C R O B I O L O G Y image and allows the viewer to access several planes of focus in the specimen (Figure 2.8). To do this, the laser beam is precisely Nucleus adjusted such that only a particular layer within a specimen is in perfect focus at one time. By precisely illuminating only this single plane, the CSLM eliminates stray light from other focal planes. Thus, when observing a relatively thick specimen such as a bacterial biofilm (Figure 2.8a), not only can cells on the surface of the biofilm be observed, as would be the case with Linda Barnett and James Barnett conventional light microscopy, but cells in the various layers can also be observed by adjusting the plane of focus of the laser beam. Using CSLM it has been possible to improve on the 0.2-mm resolution of the compound light microscope to a limit of about 0.1 mm. Cells in CSLM preparations can be stained with fluorescent Figure 2.7 Differential interference contrast microscopy. Cells of the yeast dyes to make them more distinct (Figure 2.8a). Alternatively, false Saccharomyces cerevisiae are given a three-dimensional effect by this form of color can be added to unstained preparations such that different microscopy. The yeast cells are about 8 mm wide. Note the clearly visible nucleus and layers in the specimen have different colors (Figure 2.8b). A CLSM compare to the bright-field image of yeast cells in Figure 2.5a. employs a computer to assemble digital images for subsequent image processing. Images obtained from the different layers can then be digitally reconstructed to yield a three-dimensional image of the entire specimen. CSLM is widely used in microbial ecology, especially for identifying specific populations of cells in a microbial habi- tat or for resolving the different components of a structured microbial community, such as a biofilm (Figure 2.8a) or a microbial mat. In general, CSLM is particularly useful any- where thick specimens need to be examined for their microbial content with depth. Subramanian Karthikeyan Electron source (a) Evacuated chamber Gernot Arp and Christian Boeker, Carl Zeiss, Jena Sample port Viewing screen (b) Figure 2.8 Confocal scanning laser microscopy. (a) Confocal image of a microbial biofilm community. The green, rod-shaped cells are Pseudomonas aeruginosa experimentally introduced into the biofilm. Cells of different colors are present at different depths in the biofilm. (b) Confocal image of a filamentous Figure 2.9 The electron microscope. This instrument encompasses both cyanobacterium growing in a soda lake. Cells are about 5 mm wide. transmission and scanning electron microscope functions.  CHAPTER 2 MICROBIAL CELL STRUCTURE AND FUNCTION 31 MINIQUIZ of the light microscope, even allowing one to view structures at What structure in eukaryotic cells is more easily seen in DIC than the molecular level (Figure 2.10). This is because the wavelength of electrons is much shorter than the wavelength of visible light, UNIT 1 in bright-field microscopy? (Hint: Compare Figures 2.5a and 2.7). Why is CSLM able to view different layers in a thick preparation and, as we have learned, wavelength affects resolution (Section 2.1). while bright-field microscopy cannot? For example, whereas the resolving power of a light microscope is about 0.2 micrometer, the resolving power of a TEM is about 0.2 nanometer, a thousandfold improvement. With such powerful 2.4 Probing Cell Structure: resolution, objects as small as individual protein and nucleic acid molecules can be visualized by transmission electron microscopy Electron Microscopy (Figure 2.10). Electron microscopes use electrons instead of visible light (pho- Unlike photons, electrons are very poor at penetrating; even a tons) to image cells and cell structures. In the electron micro- single cell is too thick to penetrate with an electron beam. Conse- scope, electromagnets function as lenses, and the whole system quently, to view the internal structure of a cell, thin sections of the operates in a vacuum (Figure 2.9). Electron microscopes are fitted cell are needed, and the sections must be stabilized and stained with cameras to allow a photograph, called an electron micrograph, with various chemicals to make them visible. A single bacterial to be taken. Two types of electron microscopy are in routine use cell, for instance, is cut into extremely thin (20–60 nm) slices, in microbiology: transmission and scanning. which are then examined individually by TEM (Figure 2.10a). To obtain sufficient contrast, the sections are treated with stains Transmission Electron Microscopy such as osmic acid, or permanganate, uranium, lanthanum, or The transmission electron microscope (TEM) is used to examine lead salts. Because these substances are composed of atoms of cells and cell structure at very high magnification and resolu- high atomic weight, they scatter electrons well and thus improve tion. The resolving power of a TEM is much greater than that contrast. If only the external features of an organism are to be Cytoplasmic Septum Cell wall DNA membrane (nucleoid) Stanley C. Holt (a) Robin Harris F. R. Turner (b) (c) Figure 2.10 Electron micrographs. (a) Micrograph of a thin section of a dividing bacterial cell, taken by transmission electron microscopy (TEM). The cell is about 0.8 mm wide. (b) TEM of negatively stained molecules of hemoglobin. Each hexagonal- shaped molecule is about 25 nanometers (nm) in diameter and consists of two doughnut-shaped rings, a total of 15 nm wide. (c) Scanning electron micrograph of bacterial cells. A single cell is about 0.75 mm wide. 32 U N I T 1 T H E F O U N D AT I O N S O F M I C R O B I O L O G Y observed, thin sections are unnecessary. Intact cells or cell com- image contains the maximum amount of scientific information ponents can be observed directly in the TEM by a technique that is available, color is often added to electron micrographs by called negative staining (Figure 2.10b). manipulating them in a computer. However, such false color does not improve resolution of a micrograph. Its primary value is to Scanning Electron Microscopy increase the artistic value of the image for public consumption For optimal three-dimensional imaging of cells, a scanning elec- in the mass media. The maximum scientific content and detail in tron microscope (SEM) is used (Figure 2.9). In scanning electron an electron micrograph is set at the moment the micrograph microscopy, the specimen is coated with a thin film of a heavy is taken, and thus false color will be used sparingly in electron metal, typically gold. An electron beam then scans back and forth micrographs in this book so as to present the micrographs in their across the specimen. Electrons scattered from the metal coating are original scientific context. collected and projected on a monitor to produce an image (Figure 2.10c). In the SEM, even fairly large specimens can be observed, MINIQUIZ and the depth of field (the portion of the image that remains in What is an electron micrograph? Why do electron micrographs sharp focus) is extremely good. A wide range of magnifications have greater resolution than light micrographs? can be obtained with the SEM, from as low as 15* up to about What type of electron microscope would be used to view a 100,000* , but only the surface of an object is typically visualized. cluster of cells? What type would be used to observe internal cell Electron micrographs taken by either TEM or SEM are origi- structure? nally taken as black-and-white images. Although the original II Cells of Bacteria and Archaea T wo features of prokaryotic cells that are immediately obvious upon microscopic examination are their shape and small size. A variety of shapes are possible, and in general, prokaryotes are known. For example, there are fat rods, thin rods, short rods, and long rods, a rod simply being a cell that is longer in one dimension than in the other. As we will see, there are even square bacteria extremely small relative to eukaryotic cells. Cell shape can be use- and star-shaped bacteria! Cell morphologies thus form a continu- ful for distinguishing different cells and undoubtedly has some um, with some shapes, such as rods, being very common, whereas ecological significance, but cell shape rarely has phylogenetic rel- others are more unusual. evance. By contrast, the typically small size of prokaryotes affects many aspects of their biology. Morphology and Biology Although cell morphology is easily determined, it is a poor pre- 2.5 Cell Morphology dictor of other properties of a cell. For example, under the micro- scope many rod-shaped Archaea look identical to rod-shaped In microbiology, the term morphology means cell shape. Several Bacteria, yet we know they are of different phylogenetic domains morphologies are known among prokaryotes, and the most com- ( Section 1.3). With very rare exceptions, it is impossible to mon ones are described by terms that are part of the essential predict the physiology, ecology, phylogeny, pathogenic potential, lexicon of the microbiologist. or virtually any other property of a prokaryotic cell by simply Major Cell Morphologies knowing its morphology. Examples of bacterial morphologies are shown in Figure 2.11. A cell Why is a cell the shape it is? Although we know something about that is spherical or ovoid in morphology is called a coccus (plural, how cell shape is controlled, we know little about why a particular cocci). A cylindrically shaped cell is called a rod or a bacillus. Some cell evolved the morphology it has. Several selective forces undoubt- rods form spiral shapes and are called spirilla. The cells of some edly help shape the morphology of a given species. Some examples prokaryotes remain together in groups or clusters after cell divi- of these might include optimization for nutrient uptake (small cells sion, and the arrangements are often characteristic. For instance, and others with high surface-to-volume ratios, such as appendaged some cocci form long chains (for example, the bacterium Strep- cells), swimming motility in viscous environments or near surfaces tococcus), others occur in three-dimensional cubes (Sarcina), and (helical- or spiral-shaped cells), gliding motility (filamentous bac- still others in grapelike clusters (Staphylococcus). teria), and so on. Morphology is not a trivial feature of a microbial A few bacterial groups are immediately recognizable by the cell but instead a genetically encoded property that maximizes unusual shapes of their individual cells. Examples include the spi- fitness of the organism for success in its particular habitat. rochetes, which are tightly coiled bacteria; appendaged bacteria, which possess extensions of their cells as long tubes or stalks; and MINIQUIZ filamentous bacteria, which form long, thin cells or chains of cells How do cocci and rods differ in morphology? (Figure 2.11). Is cell morphology a good predictor of other properties of the The cell morphologies described here should only be consid- cell? ered representative; many variations of these morphologies are  CHAPTER 2 MICROBIAL CELL STRUCTURE AND FUNCTION 33 2.6 Cell Size and the Significance of Being Small UNIT 1 Prokaryotes vary in size from cells as small as about 0.2 mm in Norbert Pfennig diameter to those more than 700 mm in diameter (Table 2.1). The vast Coccus majority of rod-shaped prokaryotes that have been cultured are between 0.5 and 4 mm wide and less than 15 mm long. But a few very large prokaryotes, such as Epulopiscium fishelsoni, are known, with cells longer than 600 mm (0.6 millimeter) (Figure 2.12). This bacterium, phylogenetically related to the endospore-forming bac- terium Clostridium and found in the gut of tropical marine fish called surgeonfish, contains multiple copies of its genome. The many copies are apparently necessary because the volume of an Norbert Pfennig Rod Epulopiscium cell is so large (Table 2.1) that a single copy of its Norbert Pfennig Spirillum Esther R. Angert, Harvard University E. Canale-Parola Spirochete (a) Norbert Pfennig Stalk Hypha Budding and appendaged bacteria Heide Schulz-Vogt T. D. Brock Filamentous bacteria (b) Figure 2.11 Cell morphologies. Beside each drawing is a phase-contrast Figure 2.12 Some very large prokaryotes. Dark-field photomicrograph of two photomicrograph of cells showing that morphology. Coccus (cell diameter in giant prokaryotes, species of Bacteria. (a) Epulopiscium fishelsoni. The rod-shaped photomicrograph, 1.5 mm); rod (1 mm); spirillum (1 mm); spirochete, (0.25 mm); cell is about 600 mm (0.6 mm) long and 75 mm wide and is shown with four cells budding (1.2 mm); filamentous (0.8 mm). All photomicrographs are of species of of the protist Paramecium (a eukaryote), each of which is about 150 mm long. Bacteria. Not all of these morphologies are known among the Archaea. (b) Thiomargarita namibiensis, a large sulfur chemolithotroph and currently the largest known prokaryote. Cell widths vary from 400 to 750 mm. 34 U N I T 1 T H E F O U N D AT I O N S O F M I C R O B I O L O G Y Table 2.1 Cell size and volume of some cells of Bacteria, from the largest to the smallest Organism Characteristics Morphology Sizea (mm) Cell volume (mm3) E. coli volumes Thiomargarita namibiensis Sulfur chemolithotroph Cocci in chains 750 200,000,000 100,000,000 Epulopiscium fishelsonia Chemoorganotroph Rods with tapered ends 80 * 600 3,000,000 1,500,000 Beggiatoa speciesa Sulfur chemolithotroph Filaments 50 * 160 1,000,000 500,000 Achromatium oxaliferum Sulfur chemolithotroph Cocci 35 * 95 80,000 40,000 Lyngbya majuscula Cyanobacterium Filaments 8 * 80 40,000 20,000 Thiovulum majus Sulfur chemolithotroph Cocci 18 3,000 1,500 Staphylothermus marinusa Hyperthermophile Cocci in irregular clusters 15 1,800 900 Magnetobacterium Magnetotactic bacterium Rods 2 * 10 30 15 bavaricum Escherichia coli Chemoorganotroph Rods 1*2 2 1 Pelagibacter ubiquea Marine chemoorganotroph Rods 0.2 * 0.5 0.014 0.007 Mycoplasma pneumoniae Pathogenic bacterium Pleomorphicb 0.2 0.005 0.0025 a Where only one number is given, this is the diameter of spherical cells. The values given are for the largest cell size observed in each species. For example, for T. namibiensis, an average cell is only about 200 mm in diameter. But on occasion, giant cells of 750 mm are observed. Likewise, an average cell of S. marinus is about 1 mm in diameter. The species of Beggiatoa here is unclear and E. fishelsoni, Magnetobacterium bavaricum, and P. ubique are not formally recognized names in taxonomy. b Mycoplasma is a bacterium that lacks a cell wall and can thus take on many shapes (pleomorphic means “many shapes”). Source: Data obtained from Schulz, H.N., and B.B. Jørgensen. 2001. Ann. Rev. Microbiol. 55: 105–137. genome would be insufficient to support its transcriptional and (Figure 2.13). As a cell increases in size, its S/V ratio decreases. To translational demands. illustrate this, consider the S/V ratio for some of the cells of dif- Cells of the largest known prokaryote, the sulfur chemolith- ferent sizes listed in Table 2.1: Pelagibacter ubique, 22; E. coli, 4.5; otroph Thiomargarita (Figure 2.12b), are even larger than those and E. fishelsoni (Figure 2.12a), 0.05. of Epulopiscium, about 750 mm in diameter; such cells are just The S/V ratio of a cell affects several aspects of its biology, visible to the naked eye. Why these cells are so large is not well including even its evolution. Because how fast a cell can grow understood, although for sulfur bacteria a large cell size may be depends, among other things, on the rate of nutrient exchange, a mechanism for storing inclusions of sulfur (an energy source). the higher S/V ratio of smaller cells supports a faster rate of nutri- It is hypothesized that the upper size limit for prokaryotic cells ent exchange per unit of cell volume compared with larger cells. results from the decreasing ability of larger and larger cells to As a result, smaller cells tend to grow faster than larger cells, and transport nutrients (their surface-to-volume ratio is very small; for a given amount of resources (nutrients available to support see the next subsection). Since the metabolic rate of a cell varies inversely with the square of its size, for very large cells, nutrient uptake would eventually limit metabolism to the point that the cell would no longer be competitive with smaller cells. r = 1 μm Very large cells are uncommon in the prokaryotic world. In r = 1 μm Surface area (4πr2 ) = 12.6 μm 2 contrast to Thiomargarita or Epulopiscium (Figure 2.12), the 4 Volume ( 3 πr3 ) = 4.2 μm 3 dimensions of an average rod-shaped prokaryote, the bacterium E. coli, for example, are about 1 * 2 mm; these dimensions are Surface typical of the cells of most prokaryotes. By contrast, eukaryotic =3 cells can be 2 to more than 600 mm in diameter, although very Volume small eukaryotes are uncommon, most being 8 mm in diameter or greater. In general, then, it can be said that prokaryotes are very r = 2 μm small cells compared with eukaryotes. r = 2 μm Surface area = 50.3 μm 2 Surface-to-Volume Ratios, Growth Rates, Volume = 33.5 μm 3 and Evolution Surface There are significant advantages to being small. Small cells have = 1.5 Volume more surface area relative to cell volume than do large cells; that is, they have a higher surface-to-volume ratio. Consider a coccus. The volume of a coccus is a function of the cube of its radius (V = 43 πr 3), while its surface area is a function of the square of Figure 2.13 Surface area and volume relationships in cells. As a cell the radius (S = 4πr 2). Therefore, the S/V ratio of a coccus is 3/r increases in size, its S /V ratio decreases.  CHAPTER 2 MICROBIAL CELL STRUCTURE AND FUNCTION 35 growth), a larger population of small cells than of large cells is nature. However, this is not true, as there are lower limits to cell possible. This in turn can affect evolution. size. If one considers the volume needed to house the essential Each time a cell divides, its chromosome replicates. As DNA is components of a free-living cell—proteins, nucleic acids, ribosomes, UNIT 1 replicated, occasional errors, called mutations, occur. Because and so on—a structure 0.1 mm in diameter or less is insufficient mutation rates appear to be about the same in all cells, large or to do the job, and structures 0.15 mm in diameter are marginal. small, the more chromosome replications that occur, the greater Thus, structures observed in natural samples that are 0.1 mm or the total number of mutations in the cell population. Mutations are even smaller and “look” like bacterial cells are almost certainly the “raw material” of evolution; the larger the pool of mutations, the not so. Despite this, some very small prokaryotic cells are known greater the evolutionary possibilities. Thus, because prokaryotic and many have been grown in the laboratory. Open ocean water, cells are quite small and are also genetically haploid (allowing muta- for example, contains 105–106 prokaryotic cells per milliliter, and tions to be expressed immediately), they have, in general, the capacity these tend to be very small cells, 0.2–0.4 mm in diameter. We will for more rapid growth and faster evolution than larger, genetically see later that many pathogenic bacteria are also very small. When diploid cells. In the latter, not only is the S/V ratio smaller, but the genomes of these pathogens are examined, they are found to the effects of a mutation in one gene can be masked by a second, be highly streamlined and missing many genes whose functions unmutated gene copy. These fundamental differences in size and are supplied to them by their hosts. genetics between prokaryotic and eukaryotic cells is a major reason why prokaryotes adapt rapidly to changing environmental condi- MINIQUIZ tions and more easily exploit new habitats than eukaryotic cells. We What physical property of cells increases as cells become will illustrate this concept in later chapters when we consider, for smaller? example, the enormous diversity of prokaryotes (Chapters 13–16) How can the small size and haploid state of prokaryotes accelerate and the rapidity of their evolution ( Section 12.6). their evolution? What are the approximate limits to how small a cell can be? Lower Limits of Cell Size Why is this so? From the foregoing, one could imagine that smaller and smaller bacteria would have greater and greater selective advantages in III The Cytoplasmic Membrane and Transport W e now consider the structure and function of one of a cell’s most critical structures, the cytoplasmic membrane. The cytoplasmic membrane plays many roles, chief among them as The cytoplasmic membrane is only 8−10 nanometers wide but is still visible in the transmission electron microscope, where it appears as two dark lines separated by a light line (Figure 2.14c). the “gatekeeper” for dissolved substances that enter and exit This unit membrane, as it is called (because each phospholipid the cell. leaf forms half of the “unit”), consists of a phospholipid bilayer with proteins embedded in it (Figure 2.15). Although in a diagram 2.7 Membrane Structure the cytoplasmic membrane appears rigid, it is actually somewhat fluid, having a consistency approximating that of a low-viscosity The cytoplasmic membrane surrounds the cytoplasm and sepa- oil. Thus, some freedom of movement exists for proteins embed- rates it from the environment. If the cytoplasmic membrane is ded in the membrane. The cytoplasmic membranes of some Bac- compromised, the integrity of the cell is destroyed, the cytoplasm teria are strengthened by sterol-like molecules called hopanoids. leaks into the environment, and the cell dies. The cytoplasmic Sterols are rigid and planar molecules that function to strengthen membrane is structurally weak and confers little protection from the membranes of eukaryotic cells, and hopanoids serve a similar osmotic lysis, but it is an ideal structure for its major function on function in Bacteria. the cell: selective permeability. Membrane Proteins Composition of Membranes The protein content of the cytoplasmic membrane is quite high, The general structure of the cytoplasmic membrane is a phos- and membrane proteins typically have hydrophobic surfaces in pholipid bilayer. Phospholipids are composed of both hydropho- regions that span the membrane and hydrophilic surfaces in regions bic (fatty acid) and hydrophilic (glycerol–phosphate) components that contact the environment and the cytoplasm (Figures 2.14 and (Figure 2.14). As phospholipids aggregate in an aqueous solution, 2.15). The outer surface of the cytoplasmic membrane faces the they naturally form bilayers. In a phospholipid membrane, the environment and in gram-negative bacteria interacts with a vari- fatty acids point inward toward each other to form a hydrophobic ety of proteins that bind substrates or process larger molecules for environment, and the hydrophilic portions remain exposed to the transport into the cell (periplasmic proteins, Section 2.11). The external environment or the cytoplasm (Figure 2.14b). Common inner surface of the cytoplasmic membrane touches the cytoplasm fatty acids in the cytoplasmic membrane include those with 14 to and interacts with proteins and other molecules in this milieu. 20 carbon atoms. 36 U N I T 1 T H E F O U N D AT I O N S O F M I C R O B I O L O G Y Glycerol The cytoplasmic membrane of Archaea is formed from either glycerol diethers, which have 20-carbon side chains (the 20-C O H unit is called a phytanyl group composed of 5 isoprene units), or C O C H H3C O diglycerol tetraethers, which have 40-carbon side chains (Figure C O C H 2.17). In the tetraether lipid, the ends of the phytanyl side chains H3C O that point inward from each glycerol molecule are covalently Fatty acids H C O P O– linked. This forms a lipid monolayer instead of a lipid bilayer H O membrane (Figure 2.17d, e). In contrast to lipid bilayers, lipid Phosphate CH2 monolayer membranes are extremely resistant to heat and are CH2 therefore widely distributed among hyperthermophilic Archaea, Ethanolamine + NH3 organisms that grow best at temperatures above 80°C. Mem- (a) branes with a mixture of bilayer and monolayer character are also possible, with some of the opposing hydrophobic groups cova- Hydrophilic lently bonded and others not. region Many archaeal lipids contain rings within the hydrocarbon side chains. For example, crenarchaeol, a lipid widespread among spe- Fatty acids Hydrophobic cies of Thaumarchaeota, a major phylum of Archaea, contains region four 5-carbon (cyclopentyl) rings and one 6-carbon (cyclohexyl) ring (Figure 2.17c). Rings in the hydrocarbon side chains affect Hydrophilic region the chemical properties of the lipids and thus overall membrane function. Sugars can also be present in archaeal lipids. For exam- (b) ple, the predominant membrane lipids of many Euryarchaeota, a Glycerophosphates major group of Archaea that includes the methanogens and ex- treme halophiles ( Figure 1.6b), are glycerol diether glycolipids. G. Wagner Fatty acids Despite the differences in chemistry between the cytoplasmic membranes of Archaea and organisms in the other domains, (c) the fundamental construction of the archaeal cytoplasmic Figure 2.14 Phospholipid bilayer membrane. (a) Structure of the phospholipid membrane—inner and outer hydrophilic surfaces and a hydropho- phosphatidylethanolamine. (b) General architecture of a bilayer membrane; the blue bic interior—is the same as that of membranes in Bacteria and spheres depict glycerol with phosphate and/or other hydrophilic groups. (c) Transmis- Eukarya. Evolution has selected this design as the best solution to sion electron micrograph of a membrane. The light inner area is the hydrophobic the main function of the cytoplasmic membrane—permeability— region of the model membrane shown in part b. and we consider this problem now. Many membrane proteins are firmly embedded in the mem- MINIQUIZ brane and are called integral membrane proteins. Other proteins Draw the basic structure of a lipid bilayer and label the hydrophilic have one portion anchored in the membrane and extramembrane and hydrophobic regions. regions that point into or out of the cell (Figure 2.15). Still other How are the membrane lipids of Bacteria and Archaea similar, proteins, called peripheral membrane proteins, are not membrane- and how do they differ? embedded but nevertheless remain associated with membrane surfaces. Some of these peripheral membrane proteins are lipo- proteins, molecules that contain a lipid tail that anchors the pro- tein into the membrane. Peripheral membrane proteins typically 2.8 Membrane Function interact with integral membrane proteins in important cellular The cytoplasmic membrane has several functions. First and fore- processes such as energy metabolism and transport. Membrane most, the membrane is a permeability barrier, preventing the pas- proteins that need to interact with each other in some process sive leakage of solutes into or out of the cell (Figure 2.18). Second, are typically grouped together into clusters to allow them to the membrane is an anchor for many proteins. Some of these are remain adjacent to one another in the semifluid environment of enzymes that function in energy conservation, and others trans- the membrane. port solutes into and out of the cell. The cytoplasmic membrane is a major site of energy conservation in the prokaryotic cell. The Archaeal Membranes membrane can exist in an energetically charged form in which In contrast to the lipids of Bacteria and Eukarya in which ester protons (H+) are separated from hydroxyl ions (OH-) across linkages bond fatty acids to glycerol, the lipids of Archaea contain the membrane surface (Figure 2.18c). Charge separation forms ether bonds between glycerol and their hydrophobic side chains an energized state, analogous to the potential energy present in (Figure 2.16). Archaeal lipids thus lack fatty acids, per se, although a charged battery. This energy source, called the proton motive the hydrophobic side chains play the same functional role as fatty force, is responsible for driving many energy-requiring functions acids. Archaeal lipids are formed from multiple units of the five- in the cell, including many transport reactions, swimming motil- carbon hydrocarbon isoprene (Figure 2.16c). ity, and the biosynthesis of ATP.  CHAPTER 2 MICROBIAL CELL STRUCTURE AND FUNCTION 37 Out UNIT 1 Phospholipids Hydrophilic groups 6–8 nm Hydrophobic groups In Integral membrane proteins Phospholipid molecule Figure 2.15 Structure of the cytoplasmic membrane. The inner surface (In) faces the cytoplasm and the outer surface (Out) faces the environment. Phospholipids compose the matrix of the cytoplasmic membrane with proteins embedded or surface associated. The general architecture of the cytoplasmic membrane is similar in both prokaryotes and eukaryotes, although there are chemical differences. Permeability can be seen, most substances cannot diffuse into the cell and thus The cytoplasm is a solution of salts, sugars, amino acids, nucleo- must be transported. tides, and many other substances. The hydrophobic portion of the One substance that does freely pass the membrane in both cytoplasmic membrane (Figures 2.14 and 2.15) is a tight barrier directions is water, a molecule that is somewhat polar but suffi- to diffusion of these substances. Although some small hydropho- ciently small to pass between phospholipid molecules in the lipid bic molecules pass the cytoplasmic membrane by diffusion, polar bilayer (Table 2.2). In addition to water that enters by diffusion, and charged molecules do not diffuse but instead must be trans- membrane proteins called aquaporins function to accelerate the ported. Even a substance as small as a proton (H+) cannot diffuse movement of water across the membrane. For example, aquaporin across the membrane. The relative permeability of the membrane AqpZ of Escherichia coli imports water to or exports water from to some biologically relevant substances is shown in Table 2.2. As the cytoplasm, depending on osmotic conditions. O Ester Ether Table 2.2 Comparative permeability of membranes to various H2C O C R O H2C O C R molecules HC O C R HC O C R Potential for diffusion O O Substance Rate of permeabilitya into a cell CH3 Water 100 Excellent H2C O P O– H2C O P O– H2C C C CH2 Glycerol 0.1 Good O– O– H Tryptophan 0.001 Fair/Poor Bacteria Archaea Eukarya Glucose 0.001 Fair/Poor (a) (b) (c) Chloride ion (Cl-) 0.000001 Very poor Potassium ion (K+) 0.0000001 Extremely poor Figure 2.16 General structure of lipids. (a) The ester linkage and (b) the ether linkage. (c) Isoprene, the parent structure of the hydrophobic side chains of archaeal Sodium ion (Na+) 0.00000001 Extremely poor lipids. By contrast, in lipids of Bacteria and Eukarya, the side chains are composed of a Relative scale—permeability with respect to permeability to water given as 100. fatty acids (see Figure 2.14a). Permeability of the membrane to water may be affected by aquaporins. 38 U N I T 1 T H E F O U N D AT I O N S O F M I C R O B I O L O G Y Phytanyl CH3 H2C O C CH3 HC O C H2COPO32– CH3 groups (a) Glycerol diether Isoprene unit Biphytanyl –2 H2C O C 3OPOCH2 C O CH HC O C H2COPO32– C O CH2 (b) Diglycerol tetraethers HOH2C HC O C H2C O C C O CH2 (c) Crenarchaeol C O CH CH2OH Out Out Glycerophosphates Phytanyl Biphytanyl or crenarchaeol Membrane protein In In (d) Lipid bilayer (e) Lipid monolayer Figure 2.17 Major lipids of Archaea and the architecture of archaeal membranes. (a, b) Note that the hydrocarbon of the lipid is bonded to the glycerol by an ether linkage in both cases. The hydrocarbon is phytanyl (C20) in part a and biphytanyl (C40) in part b. (c) A major lipid of Thaumarchaeota is crenarchaeol, a lipid containing 5- and 6-carbon rings. (d, e) Membrane structure in Archaea may be bilayer or monolayer (or a mix of both). Transport Proteins to high concentration, and as we will see in the next section, this Transport proteins do more than just carry solutes across the costs the cell energy. membrane—they function to accumulate solutes against the con- Transport systems show several characteristic properties. First, centration gradient. The necessity for carrier-mediated transport in contrast with diffusion, transport systems show a saturation is easy to understand. If diffusion were the only mechanism by effect. If the concentration of a substrate is high enough to sat- which solutes entered a cell, the intracellular concentration of urate the transporter, which often occurs at very low substrate nutrients would never exceed that of the external concentration, concentrations, the rate of uptake becomes maximal and the ad- which for most nutrients in nature is often quite low (Figure 2.19). dition of more substrate does not increase the rate (Figure 2.19). This would be insufficient for cells to carry out biochemical reac- This feature of transport proteins is essential for concentrating tions. Transport reactions move nutrients from low concentration nutrients from very dilute environments. A second characteristic  CHAPTER 2 MICROBIAL CELL STRUCTURE AND FUNCTION 39 Functions of the cytoplasmic membrane + ++ + + + + + + + + + + + + + + + + + UNIT 1 + – – – – – – – – – – – – – – – – –– + + –– – + + – – – + – + – OH - – + + –– – – – – – – – – – – –– + ++ + ++ + + + + + + + + + + + + + + + + H (a) Permeability barrier: (b) Protein anchor: (c) Energy conservation: Prevents leakage and functions as a Site of many proteins that participate in Site of generation and dissipation of the gateway for transport of nutrients into, transport, bioenergetics, and chemotaxis proton motive force and wastes out of, the cell Figure 2.18 The major functions of the cytoplasmic membrane. Although structurally weak, the cytoplasmic membrane has many important cellular functions. of carrier-mediated transport is high specificity. Many transport requirements, several transport mechanisms exist in prokaryotes, proteins carry only a single kind of molecule, whereas a few carry each with its own unique features. a related class of molecules, such as several different sugars or several different amino acids. This economizing reduces the need Transport Events and Transporters for separate transport proteins for each different amino acid or At least three transport mechanisms have been well characterized sugar. A third major characteristic of transport systems is that in prokaryotes. Simple transport consists only of a membrane- their synthesis is often highly regulated by the cell. That is, the spanning transport protein, group translocation employs a series specific complement of transporters present in the cytoplasmic of proteins in the transport event, and ABC transport systems con- membrane of a cell is a function of both the nature and concen- sist of three components: a substrate-binding protein, a membrane- tration of resources in its environment. Some nutrients are trans- integrated transporter, and an ATP-hydrolyzing protein (Figure ported by one transporter when present at high concentration 2.20). All of these systems drive the actual transport event using and by a separate, usually higher-affinity transporter, when pres- the energy of the proton motive force or ATP or some other energy- ent at very low concentration. rich organic compound. Membrane transporters are typically composed of 12 polypep- MINIQUIZ tides that weave back and forth through the membrane to form a Why can a cell not depend on simple diffusion as a means of acquiring its nutrients? Simple transport: Out In Why is physical damage to the cytoplasmic membrane potentially Driven by the energy in the proton motive lethal for the cell? H+ force H+ 2.9 Nutrient Transport Transported To fuel metabolism and support growth, cells need to import nu- substance trients and export wastes on a continuous basis. To fulfill these