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70 CHAPTER 4 The fungal microcolony is growing on a desert rock. The X-ray data for the fungus reveal the presence of manganese, iron, and other elements. at both the organismal and the subcellular level, their chemical composition, and their functional role in the organism. Keep in mind that mos...

70 CHAPTER 4 The fungal microcolony is growing on a desert rock. The X-ray data for the fungus reveal the presence of manganese, iron, and other elements. at both the organismal and the subcellular level, their chemical composition, and their functional role in the organism. Keep in mind that most microbiologists study the behavior of microorganisms in the laboratory, although their ultimate interest lies in understanding the function of a given structure in the organism’s natural environment. The X-ray analyses of four areas of surrounding rock reveal iron and other elements but no manganese. MORPHOLOGY OF BACTERIA AND ARCHAEA Bacteria and Archaea come in a variety of simple shapes. Most are single-celled but some are multicellular forms consisting of numerous cells living together. Figure 4.14 SEM-elemental analysis SEM of a fungal microcolony growing on a desert rock. The X-ray findings suggest that the colony is accumulating manganese. Courtesy of F. Palmer and J. T. Staley. (A) (B) Unicellular Organisms The simplest shape for a single-celled prokaryote is the sphere (Figure 4.15). Unicellular spherical organisms are called cocci. Some cocci (E) (C) (D) Figure 4.15 Cocci Various formations of cocci, spherical cells, as shown by microscopy. (A) staphylococcus, a grapelike cluster. (B) diplococcus, a pair of cells; (C) sheet (internal bright areas of each cell are gas vacuoles); (D) eight-cell packet, or sarcina; (E) streptococcus, a chain of cells. A, © Dennis Kunkel Microscopy, Inc.; B, © M. Abbey/Visuals Unlimited; C,D, courtesy of J. T. Staley and J. Dalmasso; E, © David M. Phillips/Visuals Unlimited. STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA grow and divide along only one axis. If the cells remain attached after cell division, this results in the formation of chains of cells of various lengths. A diplococcus is a “chain” of only two cells (Figure 4.15B); a streptococcus can contain many cells in its chain (Figure 4.15E). Some cocci divide along two perpendicular axes in a regular fashion to produce a sheet of cells (Figure 4.15C). Other cocci divide along three perpendicular axes, resulting in the formation of a packet or sarcina of cells (Figure 4.15D). Finally, random division of a coccus produces a grapelike cluster of cells referred to as a staphylococcus (Figure 4.15A). The most common shape in the prokaryotic world is not a sphere, however. It is a cylinder with blunt ends, referred to as a rod or bacillus (Figure 4.16). Some rods remain attached to one another after division across the transverse (short) axis of the cell, forming a chain. A less common shape for unicellular bacteria is a helix. A very short helix (less than one helical wavelength long) is called a bent rod or vibrio (Figure 4.17A). A longer helical cell is called a spirillum (Figure 4.17B) if the cell shape is rigid and unbending or a spirochete (Figure 4.17C) if the organism is flexible and changes its shape during movement. Variations of these common shapes of unicellular bacteria also exist. For example, some bacteria produce appendages that are actually extensions of the cell, called prosthecae, which give the cell a star-shaped appearance (Figure 4.18). The morphological diversity of Bacteria and Archaea is described further in Chapters 18 to 22, where individual genera are discussed. 71 (A) (B) Multicellular Prokaryotes Numerous prokaryotic organisms exist as multicellular forms. One such group is the actinobacteria. These rod(C) Figure 4.17 Curved and helical cells Figure 4.16 Bacilli, or rods Rod-shaped cell of a unicellular Bacillus anthracis, shown by phase contrast microscopy. ©Dennis Kunkel Microscopy, Inc. (A) Bent rod, or vibrio; (B) spirillum; (C) large spirochete, from the style of an oyster. A, B, ©Dennis Kunkel Microscopy Inc.; C, ©Paul W. Johnson and John Sieburth/Biological Photo Service. 72 CHAPTER 4 (A) Figure 4.18 Prosthecate bacterium A star-shaped bacterium, Ancalomicrobium adetum. Courtesy of J. T. Staley. shaped organisms produce long filaments containing many cells. The filaments form branches, resulting in an extensive network comprising hundreds or thousands of cells. This network is referred to as a mycelium (Figure 4.19). Another common multicellular shape is the trichome, which is frequently encountered in the cyanobacteria (Figure 4.20A). Although a trichome superficially resembles a chain, adjoining cells have a much closer spatial and physiological relationship than do the cells in a chain. Motility and other functions result from the concerted action of all cells of the trichome. And some cells in the trichome may have specialized functions that benefit the entire trichome. For example, the heterocyst (Figure 4.20B), seen in some filamentous cyanobacteria, is the site of nitrogen fixation (see Chapter 21). (B) Heterocyst Akinete Figure 4.20 Filamentous bacteria (A) Oscillatoria, a multicellular filamentous cyanobacterium, showing the close contact between cells in the trichome. (B) An Anabaena sp. filament with typical and specialized cells. Heterocysts are nonpigmented cells, the site of nitrogen fixation; the akinete is a resting stage. A, ©James W. Richardson/ Visuals Unlimited; B, ©Paul W. Johnson/Biological Photo Service. CELL DIVISION OF BACTERIA AND ARCHAEA Prokaryotes maintain their shapes during the process of asexual reproduction—a process in which a single organism divides to produce two progeny. For unicellular prokaryotes there are two ways in which this may be accomplished, either by binary transverse fission or by budding. Binary Transverse Fission Figure 4.19 Mycelial bacterium Streptomyces sp. illustrating the complex network of filaments called a mycelium. Courtesy of J. T. Staley and J. Dalmasso. The most common type of bacterial cell division is binary transverse fission. In this process, the cell (which may be a coccus, rod, spirillum, or other shape) elongates as growth occurs along its longitudinal axis (Figure 4.21). When a certain length is reached, a septum (wall structure) is produced along the transverse axis of the cell midway between the cell ends. When the septum has completely formed, the two resulting cells become separate entities. This process is called binary STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA (A) (B) Figure 4.21 Binary transverse fission Typical binary transverse fission in (A) a rod-shaped bacterium and (B) a coccus. fission because two cells are produced by a division or “splitting” of one original cell. The process is described as transverse because the septum that separates the two new cells is formed along the transverse, or short, axis of the original cell. In binary transverse fission, DNA replication precedes septum formation. The two resulting cells are mirror images of one another. Analyses of cell wall components of dividing cells indicate that the chemical constituents of the original “mother” cell wall are equally shared in the cell walls of the two “daughter” cells. In multicellular prokaryotes with trichomes, the organism divides by transverse fission and the trichome ultimately separates into two separate trichomes. Budding Budding, or bud formation, is a less common form of cell division among prokaryotic organisms. As in binary transverse fission, this is an asexual division process that 1 A small protuberance (bud) forms on the mother cell and enlarges as growth proceeds. results in the formation of two cells from the original cell. In the budding process, however, a small protuberance, a bud, is formed on the cell surface. The protuberance enlarges as growth proceeds and eventually becomes sufficiently large and mature to separate from the mother cell (Figure 4.22) Binary transverse fission and budding differ in several ways. During binary transverse fission, the symmetry of the cell with respect to the longitudinal and transverse axes is maintained throughout the entire process (Figure 4.21). This results in the mother cell producing two daughter cells and losing its identity in the process. In the budding process, however, symmetry with respect to the transverse axis is not maintained during division (Figure 4.22). Also, in contrast to binary transverse fission, most of the new cell wall components are used in the synthesis of the bud instead of being divided equally between the two progeny cells. The result is that, during budding, the mother cell produces one daughter cell while retaining its identity generation after generation. Whether there is a limit to the number of buds a mother cell can produce during its existence is as yet unknown. Fragmentation Another type of cell division process occurs in the mycelial bacterial group, the actinobacteria or streptomycetes. These organisms have “multinucleate” filaments that lack septa between the cells; these filaments are referred to as coenocytic and are analogous to the filaments found in some fungi. Some bacteria of this type undergo a multiple fission process. Actinobacteria undergo a fragmentation in which the filament develops septations between the nuclear areas, resulting in the simultaneous formation of numerous unicellular rods. In an analogous fashion, some cyanobacteria produce numerous smaller daughter cells called baeocytes from a single large mother cell during cell division (see Chapter 21). 2 When the bud is sufficiently mature, it separates from the mother cell. Figure 4.22 Budding Bud formation in Ancalomicrobium adetum, a prosthecate bacterium. The budding Perry /can Staley Lory process be repeated as long as nutrients are available for growth. Microbiology 2/e, Sinauer Associates Figure 04 22 Date 02/14/02 73 3 The mother cell produces another bud from the same location on the cell surface and daughter cell produces first bud. 74 CHAPTER 4 Methods & Techniques Box 4.2 In order to determine the composition of various components of the cell, scientists separate these components from the rest of the cell, purify them, and analyze them biochemically.The initial step is to break open the cells (see figure). Either chemical or physical procedures can be used to break open small, prokaryotic cells. For example, chemical procedures include lysis of the cells by enzymes or detergents. Physical methods include ultrasound (called sonication by biologists), in which high-frequency sound waves vibrate cells until they break. A sonicator probe is inserted into a cell suspension for this purpose, as shown in the illustration. Alternatively, cells can be broken by passing thick suspensions of frozen cells through a small orifice (French pressure cell) at high pressure. Cell Fractionation, Separation, and Biochemical Analyses of Cell Structures Once the cells have been broken, the various structural fractions are separated, usually by centrifugation. Two types of centrifugation can be used. In differential or velocity centrifugation, fractions are separated by the length of time they are centrifuged at different gravitational forces. Denser structures such as unbroken cells or bacterial endospores, cell membranes, or cell walls sediment at low speeds (15,000 × g for 10 minutes).The supernatant is removed and centrifuged at higher speed to spin out less dense structures. For example, ribosomes sediment only after centrifugation at higher speeds (100,000 × g for 60 minutes).The remaining material that does not sediment in the centrifuge tube contains soluble constituents such as cytoplasmic enzymes. FINE STRUCTURE, COMPOSITION, AND FUNCTION IN BACTERIA AND ARCHAEA The remainder of this chapter is devoted to the description of various prokaryotic structures, their chemical composition, and their functions. The terms “fine structure” and “ultrastructure” refer to subcellular features that are best observed using the electron microscope. Studies of the fine structure of microbial cells began in the 1950s and 1960s, when electron microscopy procedures were perfected. Scientists used a combination of procedures to “break open” or lyse cells, followed by centrifugation to separate the various subcellular components. These components were purified and then analyzed biochemically. The electron microscope was used at various steps in the procedure to identify and assess the purity of the structures (Box 4.2). We begin with internal structures found in the cytoplasm and then consider the outer layers of the cell. The discussion in this chapter is confined to structures commonly found in many prokaryotic phyla. Thus, we do not discuss structures such as spores and magnetite crystals, which are covered in descriptions of the particular phyla that have these structures in Chapters 18 through 22. Alternatively, buoyant density or density gradient centrifugation can be used to separate the various cell fractions. In this procedure a density gradient is set up in the centrifuge using different concentrations of a solute, such as sucrose.The sample is layered on the surface and centrifuged at moderate speed until the cellular fractions equilibrate with the layer in the gradient that has the same buoyant density.They can then be removed with a pipette and studied as purified fractions. When the cell fraction that is of interest to the microbiologist is separated and purified by the procedures outlined above, it can be analyzed chemically.The electron microscope is used to check the identity and purity of the material at each step in the process. Internal Structures Foremost among the intracellular materials of all cells is their DNA, the hereditary material of the cell. DNA and other intracellular components commonly found in many different Bacteria and Archaea, including ribosomes, gas vesicles, and various reserve materials, are discussed individually below. DNA The deoxyribonucleic acid (DNA) of prokaryotes is a circular, or more rarely a linear, double-stranded helical molecule. The two strands are held together by hydrogen bonds, the nucleotide bases of one strand forming hydrogen bonds with the bases of the opposite strand: adenine with thymine and cytosine with guanine (see Chapter 3). The DNA appears as a fibrous material in the cytoplasm when prokaryotic cells are viewed in thin sections (Figure 4.23). As noted in Chapter 1, the DNA of prokaryotes is not surrounded by a membrane and thus does not appear in a confined area within the cell; rather, it appears as a somewhat diffuse, dispersed fibrous material. For this reason the region is not called a nucleus but a nucleoid or nuclear area. If gentle conditions are used to lyse the cells (like eggs, cells can be broken carefully; the cytoplasm can be freed STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA Methods & Techniques Box 4.2 Cell suspension 75 (continued) 1 Disrupt cells by sonication. Sonicator causes cell breakage by producing high-frequency sound waves Approach a: Differential centrifugation 2a Centrifuge the cell suspension at low speed (15,000 × g for 10 minutes), then examine the sediment in the electron microscope to confirm identity and purity. If pure, analyze biochemically. Approach b: Bouyant density centrifugation Cell suspension Supernatant Cell walls, membranes, flagella A density gradient of sucrose 3b Centrifuge the tube to equilibrium. 3a Remove the supernatant and centrifuge again at high speed (100,000 × g for 60 minutes). 4a Examine the sediment in the electron microscope and, if pure, analyze biochemically. Supernatant (soluble proteins, enzymes) Ribosomes, membranes, fragments from the cell membrane and wall), the DNA is released and appears as a coiled structure spilled from the cell (Figure 4.24). When stretched out, the length of the DNA molecule is about 1 mm, about a 1,000 times longer than the 1 to 3 µm length of the typical prokaryotic cell! In order to package all of this material within the cell, the DNA molecule is tightly wound in supercoils (Figure 4.25). Special enzymes are responsible for supercoiling and for controlling the unwinding of the DNA during replication (DNA synthesis) and transcription (production of RNA from the DNA template; see Chapter 13). The molecular weight of the DNA molecule of prokaryotes ranges from about 109 to 1010 Da (Da is a dalton, a unit of mass approximately equal to the mass of the hydrogen atom, 1H). The typical prokaryotic 2b 5 Layer the suspension of disrupted cells on a sucrose density gradient (with increasing density toward the bottom of the tube). Cell fractions separated at different buoyant densities 4b Examine the cell fractions in the electron microscope to confirm identity and purity. If pure, analyze biochemically. DNA contains about 4 × 106 base pairs (4 mega-base pairs, or 4 mgb). However, some intracellular symbiotic bacteria such as Buchnera species have smaller genomes (0.65 mgb), and some prokaryotic genomes are as large as 10 mgb. This is considerably smaller than the size of eukaryotic genomes, whose chromosomes may be ten times or more larger than prokaryotic chromosomes, but larger than those of viruses. Prokaryotic cells may have more than one copy of the DNA molecule. For example, when the cell is growing and dividing rapidly, two or four or more copies, or partial copies, may be present. In addition to the genomic DNA, the cell often contains other, extrachromosomal circular molecules of DNA called plasmids. These, too, are double-stranded 76 CHAPTER 4 Covalently closed circular duplex (supercoiled) N Figure 4.25 Supercoiled DNA Figure 4.23 Appearance of DNA by electron microscopy Thin section through Salmonella typhimurium showing the fibrous appearance and diffuse distribution of the nuclear material (N) in a typical prokaryotic cell. Courtesy of Stuart Pankratz. Increasing degrees of supercoiling of DNA produce a tightly compacted molecule. DNA molecules (see Chapter 15). Plasmids do not carry genetic material that is essential to the growth of an organism, although they do contain features that may enhance the survivability of the organism in a particular environment. For example, some bacteria carry a plasmid with genes that allow them to degrade naphthalene—which is not useful to the bacterium unless naphthalene is in its immediate environment. The primary function of the prokaryotic genome is to store its hereditary information, carried in its genes. Furthermore, in some prokaryotes, under the appropriate conditions genetic material can be transferred from one organism to another. This can be accomplished by three different processes, depending on the prokaryote (see Chapter 15): • Transformation, occurring when DNA released into the environment by lysis (cell breakage) of one organism is taken up by another organism • Conjugation, in which transfer occurs during cell to cell contact between two closely related bacterial strains • Transduction, in which prokaryotic viruses are involved in transferring DNA from one organism to another Ribosomes As mentioned in Chapter 1, ribosomes are Figure 4.24 DNA strands released from cell Photomicrograph showing DNA strands released from a lysed bacterial cell. ©Dr. Gopal Murti/SPL/Science Source/ Photo Researchers Inc. small structures that carry out protein synthesis (Figure 4.26), a process referred to as translation (see Chapter 13) in which messenger RNA (mRNA) carries the message in nucleotides from the genome to the ribosome, where amino acids are linked together by peptide bonds to form protein. At high magnification the prokaryotic ribosome can be seen to consist of two subunits, the small 30S subunit and the larger 50S subunit (Figure 4.27). Note that the Svedberg units (S), sedimentation densities, are not STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA Ribosomes are attached as polyribosomes to the mRNA and are translating it into protein. The DNA molecule is being transcribed to form mRNA. Figure 4.26 Protein synthesis Ribosomes in action in E. coli. The DNA and RNA appear as filaments. Courtesy of Oscar L. Miller. (A) (B) Prokaryotic ribosome (Escherichia coli) 70S Front view The 30S subunit consists of 16S rRNA (1,542 nucleotides), and 21 proteins; its mass is 0.93 × 106 Da. Side view Ribosome 30S 50S Subunits (C) Eukaryotic ribosome (Rat) 80S 77 additive: the 30S and 50S ribosome subunits comprise a 70S ribosome. This is because the Svedberg unit is directly related not to molecular mass but to the density of particles in ultracentrifugation. (Likewise, the eukaryotic 80S ribosome consists of a small 40S subunit and a larger 60S subunit.) Ribosomes consist of both protein and a type of ribonucleic acid called ribosomal RNA (rRNA). Figure 4.27 (B and C) shows the RNA and protein components of each of these subunits from the 70S and 80S ribosomes. Even though bacterial and archaeal ribosomes have the same sedimentation coefficients, they differ somewhat in structure and composition. As a result, these organisms respond differently to the same antibiotic (a substance produced by one organism that inhibits or kills other organisms). For example, certain antibiotics, including chloramphenicol and the aminoglycosides, disrupt ribosome activity (and therefore inhibit protein synthesis) in Bacteria, but have no adverse effect on Archaea. Likewise, eukaryotic ribosomes are not sensitive to some of the antibiotics that affect bacteria. Gas Vesicles Among the most unusual structures found in prokaryotes are gas vesicles, produced by some aquatic species. These special protein-shelled structures provide buoyancy to many aquatic prokaryotes, and they are not found in any other life forms. When bacteria with gas vesicles are observed in the phase microscope they appear to contain bright, refractile areas, called gas vacuoles, with an irregular outline (Figure 4.28A). When gas vacuolate cells are The prokaryotic, 70S ribosome has a mass of viewed with the TEM, the vac6 2.52 × 10 Da. It has two uoles are found to consist of subunits, 30S and 50S. numerous subunits, called gas vesicles (Figure 4.28B). Gas vacuoles are found in widely disparate prokaryotes. The 50S subunit consists of 23S rRNA (2,904 nucleotides), They are common in photosyn5S rRNA (120 nucleotides), thetic groups such as the cyanand 31 proteins; its mass is obacteria, proteobacteria, and 1.59 × 106 Da. green sulfur bacteria (Chlorobi). They are also found in hetThe eukaryotic, 80S erotrophic bacteria such as the ribosome has a mass of genus Ancylobacter and in the 4.22 × 106 Da. It has two subunits, 40S and 60S. Figure 4.27 Ribosome structure Ribosome The 40S subunit consists of 18S rRNA (1,874 nucleotides), and 33 proteins; its mass is 1.4 × 106 Da. 40S Subunits 60S The 60S subunit consists of 28S rRNA (4,718 nucleotides), 5.8S rRNA (160 nucleotides), and 49 proteins; its mass is 2.82 × 106 Da. (A) High-resolution electron micrograph of 70S ribosomes. (B) Composition of the Escherichia coli ribosome. (C) Composition of a eukaryotic (rat) ribosome. Photo courtesy of James Lake. 78 CHAPTER 4 (A) (B) 2.0 µm 1.0 µm Figure 4.28 Gas vacuoles and gas vesicles Gas vacuoles and gas vesicles in Ancylobacter aquaticus, a vibrioid bacterium. (A) Phase photomicrograph showing gas vacuoles and (B) electron micrograph showing the numerous transparent gas vesicles that comprise a gas vacuole. Courtesy of M. van Ert and J. T. Staley. prosthecate genera Prosthecomicrobium and Ancalomicrobium. The anaerobic gram-positive genus Clostridium also has gas vacuolate strains. Finally, some archaea produce gas vacuoles, including members of the genera Methanosarcina (methanogens) and Halobacterium. Gas vesicles have been isolated from bacteria and studied biochemically. They are obtained by gently lysing cells to release the vesicles, then separating the vesicles from cellular material by low-speed differential centrifugation (Box 4.2). Cell material is relatively dense and is spun to the bottom of the centrifuge tube, while the buoyant gas vesicles float to the surface. The purified vesicles have a distinctive shape. They appear as cylindrical structures with conical end pieces (Figure 4.29). The size varies from about 30 nm in diameter in some species to about 300 nm in others. The vesicles can exceed 1,000 nm in length, depending upon the organism. Each vesicle consists of a thin (about 2 nm thick) protein shell that surrounds a hollow space. The shell is composed of one predominant protein whose repeating subunits have a molecular mass of about 7,500 Da. Amino acid analyses of this protein from different prokaryotes indicate that its composition is highly uniform from one organism to another. About half of the protein consists of hydrophobic amino acids (such as alanine, valine, leucine, and isoleucine). It is thought that the hydrophobic amino acids are located on the inside of the shell and that their presence prevents water from entering the vesicle. Gases freely diffuse through the shell and are thus the sole constituents of the interior. Gas vesicles do not store gases in the same way as a balloon. Gases freely diffuse through the vesicle shell, and the structure maintains its shape not because it is inflated but because of its water-impermeable, rigid protein framework. Because all gases freely diffuse in and out of the gas vesicle, the gases found in the vesicles are those present in the organism’s environment. The primary function of gas vesicles is to provide buoyancy for aquatic prokaryotes (Box 4.3). The density of the organism is reduced when the cell contains gas vesicles, thus permitting the organism to be buoyant in aquatic habitats. The mechanisms by which organisms regulate gas vacuole formation in nature are only poorly understood. Some cyanobacteria can descend in the environment by producing increased quantities of dense storage materials such as polysaccharides or by collapsing weaker gas vesicles. Depending upon their requirements for light, oxygen, and hydrogen sulfide, various bacterial groups are found in different strata in lakes during summer thermal stratification (see Chapter 24). Figure 4.29 Gas vesicles Gas vesicles isolated from Ancylobacter aquaticus. The vesicle diameter is about 0.1 µm. Courtesy of J. T. Staley and A. E. Konopka. STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA Milestones Box 4.3 The Hammer, Cork, and Bottle Experiment In the early 1900s, C. Klebahn conducted an important but simple experiment called the “hammer, cork, and bottle”experiment, which provided the first evidence that the gas vacuoles of cyanobacteria actually contain gas. For the experiment, cyanobacteria containing the purported gas vacuoles were taken from a bloom in a lake. Microscopic examination showed that the organisms contained bright areas indicative of gas vacuoles. Samples were placed into two bottles, one to be used as a control for the experiment. In both bottles, the cyanobacteria initially floated to the surface. A cork was then placed in one bottle and secured in such a way that no air space was left between the cork and the water.Then, a hammer was used to strike a sharp blow on the suspension of cyanobacteria in the corked bottle.The blow briefly increased the hydrostatic pressure of the water in the experimental bottle. The cyanobacteria in the experimental bottle soon sank to the bottom, whereas those in the control bottle remained floating on top. Furthermore, an air space formed Figure A Two bottles containing gas vacuolate cyanobacteria collected from a lake. The cell suspension with collapsed gas vesicles has a darker appearance. Gas vacuolate cells cause much greater refraction of light, so the bottle on the right appears more turbid. Figure B Appearance of the bottles after a few minutes. Courtesy of A. E. Walsby. between the water and the cork in the experimental bottle.When the cells from this bottle were subsequently examined in the microscope, no bright areas remained in the cells, showing that the cyanobacteria had lost their buoyancy and their gas vacuoles at the same time.The air space that had Intracellular Reserve Materials Prokaryotes store a variety of organic and inorganic materials as nutrient reserves. Almost all these materials are stored as polymers, thereby maintaining the internal osmotic pressure at a low level (see the discussion of osmotic pressure in “Function of the Cell Wall” below). The main organic compounds stored by bacteria are: Glycogen Starch Poly-β-hydroxybutyric acid Cyanophycin (B) (A) The vertical position that gas vacuolate species occupy in the lake depends on their cell density, which is determined by the proportion of cell volume occupied by gas vesicles at a particular time. • • • • 79 collected in the experimental bottle was due to the gas released from the broken vesicles and contained by the cork. This simple experiment showed that, indeed, gas vacuoles do contain gas and that they are essential in providing buoyancy to the cyanobacteria. Glycogen and starch are common storage materials in prokaryotic organisms. They are polymers of glucose units linked together primarily by α-1,4 linkages (see Chapter 11).These storage materials cannot be seen using a light microscope, but when observed with the electron microscope they appear as either small, uniform granules, as in some cyanobacteria (see Figure 21.16), or as larger spheroidal structures, as in heterotrophic bacteria. Glycogen and starch can be degraded as energy and carbon sources. Interestingly, glycogen is also formed by animal cells, and starch is formed by eukaryotic algae and higher plants. Unlike glycogen and starch, poly-b-hydroxybutyric acid (PHB) and related polymeric acids appear as visible granules in bacteria when viewed with a light microscope. In phase microscopy, these lipid substances appear as bright, refractile, spherical granules (Figure 80 CHAPTER 4 Figure 4.31 Polyphosphate granules Figure 4.30 PHB granules Thin section of a bacterial cell (Pseudomonas aeroginesa) shows polyphosphate granules as dark areas. ©T. J. Beveridge/Visuals Unlimited. Poly-β-hydroxybutyrate (PHB) granules appear as bright refractile areas by phase microscopy, as seen in Azotobacter sp. Courtesy of J. T. Staley. 4.30). PHB granules are usually synthesized during periods of low nitrogen availability in environments that have excess utilizable organic carbon. In contrast to glycogen and starch, PHB is not found in eukaryotic organisms. PHB is a polymer of β-hydroxybutyric acid and is synthesized from acetyl-coenzyme A (see Chapter 11).The granules are enclosed within a protein membrane, which may be responsible for their synthesis or degradation, or both. The only organic nitrogen polymer stored by prokaryotes is cyanophycin. As implied by the name, these granules occur only in cyanobacteria. They consist of a copolymer of aspartic acid and arginine and have a distinctive appearance when viewed in thin section with an electron microscope (see Figure 21.16). Prokaryotes also store a variety of inorganic compounds, including polyphosphates and sulfur. Volutin, or metachromatic granules, consists of linear polyphosphate molecules, polymers of covalently linked phosphate units. Volutin appears as dark granules in phase microscopy. The granules can be stained with methylene blue, which results in a red color caused by the metachromatic effect of the dye-polyphosphate complex. They appear as dense areas when viewed by the electron microscope (Figure 4.31), and they may volatilize under the electron beam, leaving a “hole” in the specimen. Volutin is synthesized by the stepwise addition of single phosphate units from ATP onto a growing chain of polyphosphate. Although its degradation has not been studied, volutin may serve as an energy source for the synthesis of ATP from ADP, at least in some bacteria, as well as a reserve material for nucleic acid and phospholipid synthesis. Volutin is apparently stored by organisms when nutrients are limiting their growth. However, some bacteria are known to store volutin during active growth when there is no nutrient limitation, an effect called luxurious phosphate uptake. Sulfur is stored as elemental sulfur by certain bacteria involved in the sulfur cycle. Sulfide-oxidizing pho- Sh S Figure 4.32 Sulfur granules Phase photomicrograph of the sulfur bacterium Thiothrix nivea showing many bright yellow and orange sulfur granules (S), arrow pointing to sheath (Sh). Courtesy of Judith Bland. STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA tosynthetic proteobacteria and colorless filamentous sulfur bacteria, both of which produce sulfur by the oxidation of sulfide, store the sulfur as granules, which appear as bright, slightly yellow spherical areas in their cells (Figure 4.32). The sulfur can be further oxidized to sulfate by both groups of organisms. Thus, the granules are transitory and serve as a source of energy (when oxidized by filamentous sulfur bacteria; see Chapter 19) or a source of electrons (for carbon dioxide fixation in photosynthetic bacteria; see Chapter 21). Cell Membranes Bacterial Cell Membranes The cell membrane or cytoplasmic membrane serves as the primary boundary of the cell’s cytoplasm. In prokaryotes, it looks like a typical cell membrane—two thin lines separated by a transparent area—when viewed in thin section by the electron microscope (Figures 4.12 and 4.23). At the molecular level, a model known as the fluid mosaic The hydrophilic glycerol phosphate moieties face away from the membrane into the aqueous environment inside and outside the cell. model best explains the structure and composition of the cell membrane (Figure 4.33). The cell membranes of bacteria are composed of phospholipids and proteins. As many as seven different phospholipids and approximately 200 proteins have been identified in the typical membrane of bacteria such as Escherichia coli. One of the phospholipids is phosphatidyl serine (Figure 4.34). This complex lipid consists of a glycerol moiety covalently bonded to two fatty acids by an ester linkage and, at its third carbon, to a phosphoserine. The result is a bipolar molecule with a hydrophobic end (the hydrocarbon chains of the fatty acids) and a hydrophilic end (the glycerol phosphatidyl serine). In an aqueous environment, phospholipid molecules form a bilayer consisting of two membrane leaflets: the hydrophobic chains, or tails, of the two fatty acids extend into the center of the bilayer and the hydrophilic portions face away from the bilayer (Figure 4.33). Outside of cell Polysaccharides Integral membrane proteins The hydrophobic tails of the phospholipid molecules in each leaflet of the membrane face inward to form a hydrophobic layer. 81 Protein molecules, with charged, globular structures, are embedded in the phospholipid bilayer through their hydrophobic segments. Figure 4.33 Bacterial cell membrane structure The fluid mosaic model of cell membrane structure. The bilayer structure consists of two phospholipid leaflets. Peripheral membrane proteins Cytoplasm 82 CHAPTER 4 Hydrophilic Hydrophobic O H2 C O C Ester linkages CH2 CH3 CH2 CH3 O H C C O O– C H2 O O P O O Glycerol C CH CH2 OH NH2 Serine Figure 4.34 Phospholipid Chemical structure of phosphatidyl serine, a typical phospholipid. The hydrocarbon side chains are fatty acids, typically containing 12 to 18, sometimes more, carbon atoms. Each is attached to glycerol by an ester linkage. Serine is attached to the phosphate moiety. (A) Cholesterol 22 21 20 12 18 11 1 2 19 23 26 25 17 27 16 13 14 9 24 15 8 10 5 3 7 HO 4 6 (B) A hopanoid from a cyanobacterium OH OH 19 21 17 11 14 35 16 29 1 2 32 22 10 R1 OH R2 3 4 Figure 4.35 Sterols and hopanoids (A) A sterol and (B) a hopanoid found in some bacterial cell membranes. The hopanoid shown here is from a cyanobacterium. R1 and R2 indicate two different hydrocarbon chains. The proteins of the cell membrane are embedded in the phospholipid bilayer (Figure 4.33). Many of the proteins serve in the transport of substances into the cell. The membrane is stabilized by divalent cations including calcium and magnesium. Sterols are known to have a stabilizing effect on cell membranes because of their planar chemical structures (Figure 4.35). However, most prokaryotic organisms do not have sterols in their cell membranes. The mycoplasmas (Tenericutes), which lack cell walls, are exceptions. They do not synthesize their membrane sterols but derive them from the environment in which they live. The only other group of prokaryotes that is known to contain sterols is the methanotrophic bacteria, which have special membranes involved in the oxidization of methane as an energy source. Some bacteria, such as the cyanobacteria, produce hopanoids, compounds that resemble sterols chemically and probably provide membrane stability (Figure 4.35). The cell membrane is the ultimate physical barrier between the cytoplasm and the external environment of the cell. It is selectively permeable to chemicals; the molecular size, charge, polarity, and chemical structure of a substance determine its permeability properties. For example, water and gases diffuse freely through cell membranes, whereas sugars and amino acids do not. These important organic compounds, which serve as substrates for growth and energy metabolism, are concentrated in the cell by special transport systems (see Part III). This selective permeability of cell membranes is also important in cell energetics, as discussed in Chapter 8. One additional function of the cell membrane is its role in the replication of DNA, which is attached to the membrane. Following replication, the two new DNA molecules are physically separated by new cell membrane synthesis. The eventual outcome is the separation of the two new nuclear structures prior to formation of the septum and cell division. Some prokaryotes contain membranes that extend into the cell itself. For example, phototrophic bacteria have internal membranes that are involved in photosynthesis. Some chemoautotrophs (also called chemolithotrophs) that oxidize ammonia and nitrite and methanotrophs, which oxidize methane, possess similar intracytoplasmic membranes. These bacteria are discussed in Chapters 19 and 21. Archaeal Cell Membranes Although their appearance in thin section is like that of bacterial cell membranes, the cell membranes of archaea have a different chemical composition. One of the major differences is that the lipid moieties of the membrane are not glycerol-linked esters but glycerollinked ethers. Furthermore, rather than fatty acids, STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA most archaea have isoprenoid side chains with repeating five-carbon units (Figure 4.36). Two patterns are found in archaeal cell membranes: • A bilayer, with two leaflets of glycerol ether-linked isoprenoids held together by their hydrophobic isoprenoid side chains (Figure 4.36A) • A monolayer, consisting of diglycerol tetraethers (Figure 4.36B) In the remainder of the chapter we consider the layers external to the cell cytoplasm, beginning with those farthest from the cytoplasm and working toward the cell membrane. Extracellular Layers Some prokaryotes produce discrete layers external to the cell wall. These are of several types, varying in structure and function: • • • • Capsules, or glycocalyxes Sheaths Slime layers Protein jackets, or S-layers If the external layer is removed from the cell, the organism does not lose its viability. Thus, they do not appear (A) Glycerol diether to be essential to the organisms that produce them, at least under some conditions of growth. Capsules Capsules constitute barriers that protect the cell from the external environment, no doubt aiding the cell in unknown ways, such as preventing virus attachment or slowing the desiccation process when the cell encounters dry conditions. In the laboratory, capsules can be removed without affecting the cells’ viability, indicating they are not always essential to survival. Nevertheless, several functions have been attributed to capsules, and under certain conditions they may be crucial in determining the survival of the organism in its natural environment. For example, the capsules of some species are important virulence factors (factors that enhance the ability of the organism to cause disease). This can be illustrated by the species Streptococcus pneumoniae, one of the bacteria responsible for bacterial pneumonia. Unencapsulated strains of this bacterium are not pathogenic, that is, they are not capable of causing disease. The reason is that the unencapsulated cells are more readily killed by phagocytes (white blood cells) in the host organism’s circulatory system. Apparently the white blood cells can ingest bacteria without capsules much more readily than those The lipid bilayer type of archaeal membrane consists of two layers of glycerol diethers. H2 C O C H2 H C O C H2 H2C O Y Ether linkages 20-carbon isoprenoid (phytane) groups Y = H atom, saccharide, or phosphorylated moiety. (B) Diglycerol tetraether H2C Glycerol CH2 O HC O CH2 H2C O H 83 Glycerol HO O CH2 CH2 O CH CH2 O CH2 Figure 4.36 Archaeal cell membrane structure Archaeal membranes are of two types: (A) a lipid bilayer, consisting of two leaflets of glycerol-linked isoprenoids, and (B) a lipid monolayer, consisting of a single layer of diglycerol tetraethers. The lipid monolayer type of archaeal membrane is made up of diglycerol tetraethers. Membrane protein 84 CHAPTER 4 with capsules. Thus, capsules may provide a means by which the bacteria evade the host defense system in their natural environment. Capsules are also important in mediating attachment of some bacteria. This is well illustrated by one of the bacteria responsible for dental cavities. In the presence of sucrose, Streptococcus mutans produces a polysaccharide capsule that permits the bacterium to attach to dental enamel. As S. mutans grows on the surface of the tooth, acid produced by fermentation of sucrose causes etching of the enamel, and a cavity may eventually develop. Capsules are diffuse structures that are often difficult to visualize without special techniques. This is well exemplified by the capsule of Streptococcus pneumoniae. Its capsule is not visible by ordinary phase microscopy or by simple staining techniques, because of the diffuse consistency of the polysaccharide that forms the capsule. A special technique has been developed to permit visualization of the capsule, called the Quellung (German for “swelling”) reaction. An antiserum (see Chapter 27) is prepared from capsular material of S. pneumoniae and is used to “stain” S. pneumoniae cells, complexing with their capsules. After staining, the bacterium appears to have grown much larger or to have swollen. In reality, the antiserum has complexed with the extensive but previously transparent capsule and made it thicker and more visible (Figure 4.37). Negative stains can also be used to stain capsules for light microscopy. These stains consist of insoluble particulate materials such as India ink. The dark particles of the stain do not penetrate the capsule, so a large unstained halo appears around the cell (Figure 4.38). Figure 4.38 Negative stain An India ink negative stain shows the capsule of a Bacillus sp. This is called a negative stain because the background around the capsule is stained, not the capsule itself. Courtesy of Carl Robinow. Most capsules are composed of polysaccharides (Table 4.1). Dextrans (polymers of glucose) and levans (polymers of fructose, also known as levulose) are common homopolymers (polymers consisting of identical subunits) found in capsules produced by lactic acid bacteria (Streptococcus and Lactobacillus spp.). However, some bacteria produce heteropolymers, containing more than one type of monomeric subunit. Hyaluronic acid is an example of a heteropolymer found in capsules; it consists of N-acetylglucosamine and glucuronic acid. Another example is the glucose-glucuronic acid polymer, also produced by some Streptococcus species. Capsules can also be composed of polypeptides. For example, some members of the genus Bacillus have capsules made of polymers that are repeating units of D-glutamic acid (Table 4.1). It is interesting to note that the D stereoisomers of amino acids are found almost exclusively in the capsules and cell walls of some bacteria. Virtually all other biologically produced amino acids are of the L stereoconfiguration. Thus, other than capsule and cell wall peptides, all proteins in bacteria (as well as in other organisms) contain L-amino acids. The poly-Dglutamic acid of Bacillus is also noteworthy because the amino acids are linked together by their γ-carboxyl group, not the α-carboxyl group as is characteristic of proteins. Sheaths Some prokaryotes, the sheathed bacteria, proFigure 4.37 Capsule stain Capsules of Streptococcus pneumoniae have been stained by the Quellung reaction. Courtesy of J. Dalmasso and J. T. Staley. duce a much more dense and highly organized external layer, or sheath. Unlike a capsule, this structure is easily discerned with a light microscope (Figure 4.39). Sheathed bacteria form filamentous chains and grow in flowing aquatic habitats such as rivers and springs. The STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA Table 4.1 85 Chemical composition of capsules from various bacteria Bacterium Capsular Material Structure of Repeating Units CH2 O H H Leuconostoc mesenteroides OH H Dextran O HO H n OH α-1,6-poly D-glucose O C Streptococcus pneumoniae Polyglucose glucuronate H H OH O H O HO H H CH2OH O O H HO H H OH Glucuronic acid OH n Glucose O C H H Streptococcus spp. Hyaluronic acid HO OH O H H H CH2OH O H O O H HO OH H H NH C O CH3 Glucuronic acid n N-acetyl glucosamine O C C Bacillus anthracis γ-poly D -glutamic acid OH H N CH2 CH2 C O n γ D-glutamic acid sheath protects the cells against disruption by turbulence in the water and ultimately against being removed from the area of the habitat that is most favorable for growth. The few chemical analyses of sheaths that have been made indicate that they are more complex than capsules, having, in addition to polysaccharides, amino sugars and amino acids. Slime Layers Some prokaryotes, such as those of the Cytophaga-Flavobacterium group, move by a type of motility referred to as gliding. This type of movement requires that the organism remain in contact with a solid substrate. Gliding bacteria produce a chemical “slime,” part of which is lost in the trail left by the organism during movement (Figure 4.40). Members of the Cytophaga 86 CHAPTER 4 Sh lase and chitinase enzymes, bound to the organism’s extracellular surface, are kept close to the substrate as well as the cell. In this manner the cell can derive nutrients more effectively. The chemical nature of the slime varies. In some species it is a polysaccharide. For example, Cytophaga hutchinsonii produces a heteropolysaccharide of arabinose, glucose, mannose, xylose, and glucuronic acid. The mechanism of gliding motility is unknown, but recent interesting research on Flavobacterium johnsoniae indicates that a special sulfur-containing lipid (sulfonolipid) found on the surface of the cell is essential for gliding in this organism. Protein Jackets External protein layers are produced by certain prokaryotes, such as the genus Spirillum. These protein jackets, also Figure 4.39 Sheathed bacteria called S-layers, are often highly textured surThe filamentous gram-negative bacterium Sphaerotilus natans has a faces (Figure 4.41). Their function is unknown, sheath (Sh). This species grows in flowing aquatic environments. Courtesy of J. T. Staley. although in some archaea they make up the only layer external to the cell membrane and therefore may serve as a cell wall. However, and Flavobacterium are noted for their ability to degrade some Spirillum species and certain cyanobacteria that substances such as cellulose and chitin and other high have these layers also have a cell wall, so S-layers canmolecular weight polysaccharides found in plant materinot serve that role in these organisms. For some al (cellulose) and insect or arthropod shells (chitin). It is prokaryotes, the S-layer serves as a site on the cell suradvantageous for organisms that degrade these materials face to which bacterial viruses adhere. for nutrition to be in physical contact with them. As the Cell Walls bacterium glides on the surface of the material, the celluThe cell wall and cell membrane are referred to as the cell envelope. The cell wall is the outermost cellular constituent of prokaryotic Slime trail Cells gliding together organisms. In contrast to extracellular layers, cell walls are essential to the microorganisms that produce them. Without this protection, the organisms could not survive in their normal habitats because the cells would lyse (break open and lose their cytoplasm; see below). Considerable variation in cell wall composition exists among prokaryotes. One special class of bacteria, the Tenericutes (commonly called the mycoplasmas) lack a cell wall entirely. Bacteria without cell walls survive because their cell membranes differ from those of typical bacteria. For example, their membranes contain sterols or other compounds (see above) that help stabilize membrane structure, and in this respect they are similar to animal cell membranes, which also lack cell walls. As mentioned in Chapter 1, prokaryotes are Figure 4.40 Gliding motility classified as either Archaea or Bacteria based on The myxobacterium Stigmatella aurantiaca produces slime trails as the evolutionary studies of 16S rRNA. In addition cells glide across an agar surface. Note that even the smallest units conto this difference in their RNAs, the two tain at least two cells gliding together. Courtesy of Hans Reichenbach. STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA 87 domains also differ in the chemistry of their cell walls. Almost all Bacteria have a chemical polymer in their cell walls called peptidoglycan, or murein; Archaea do not, although some have a pseudomurein (see Chapter 18). Peptidoglycan is a complex polymeric substance containing amino sugars and amino acids, as described in detail below. S-layer Bacterial Cell Wall The peptidoglycan cell wall structure varies from one bacterial species to another. However, all have the same general chemical composition. Two amino sugars, glucosamine and muramic acid, are joined together by β-1,4 linkages to form a chain, or linear polymer (a glycan). The chains of amino sugars are cross-linked by peptides. Figure 4.42 shows the peptidoglycan of Staphylococcus aureus. Note that the amino sugars are N-acetylglucosamine and N-acetylmuramic acid. Almost all bacteria have these N-substituted acetyl groups; some Bacillus species have unsubstituted amino groups on the sugars, and mycobacteria and some of the streptomycetes have N-glycolyl groups. The amino Figure 4.41 S-layers Electron micrograph of negatively stained fragments of a highly textured protein jacket, or S-layer, from Aquaspirillum serpens strain VHA. The dark areas show the highly organized nature of the structure. Courtesy of S. F. Koval. Bond broken by lysozyme The backbone of the peptidoglycan consists of chains of amino sugars, alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) molecules linked by β-1,4 glycosidic bonds. NAG Glycan chain a CH2OH O O NAM OH CH2OH O O NAG NH CH2OH C O O NH O NH C Peptide cross-links hold together the glycans, the chains of amino sugars. CH3 Lysine (a diamino acid), by forming two peptide bonds, interlinks the peptides to form the peptide bridges. O HC C CH2OH CH3 OH C O Glycan chain b CH2OH CH3 O O NH CH3 OH C O NH D-Glutamate C L-Lysine D-Alanine O NAG O L-Alanine The pentaglycine link distinguishes the gram-positive from the gram-negative peptidoglycan. NAM O CH3 L-Glysine CH3 O O L-Glysine H C CH3 C O L-Alanine L-Glysine L-Glysine L-Glysine D-Glutamate L-Lysine D-Alanine Figure 4.42 Peptidoglycan of a gram-positive bacterium Chemical structure of the peptidoglycan layer of Staphylococcus aureus. Note that some Perryamino / Staleyacids Loryin the peptide cross-links, alanine and glutamic acid, are in the 2/e, SinauerSee Associates DMicrobiology -stereoconfiguration. also Figure 4.44. O Each peptide is connected to the glycan chain by a peptide bond between the carboxyl group of the lactic acid moiety of muramic acid and the amino group of the first amino acid in the peptide. 88 CHAPTER 4 O H2N H2N H C CH2 CH CH2 CH2 CH2 CH2 CH2 CH2 C NH2 C O OH Lysine H OH wall peptidoglycan structures are known. However, certain amino acids are never found in the peptide bridge, including the sulfur-containing amino acids, aromatic amino acids, and branched-chain amino acids, as well as arginine, proline, and histidine (see Chapter 3). Gram-Positive and Gram-Negative Bacterial Cell Walls The domain Bacteria is divided into two groups C NH2 C O OH Diaminopimelic acid (DAP) Figure 4.43 Diamino acids Lysine and diaminopimelic acid are diamino acids found in peptidoglycans. based upon the cells’ reaction to a staining procedure called the Gram stain (Box 4.4). The differences between gram-positive and gram-negative bacteria relate to differences in their cell wall structure and chemical composition. Thin sections of gram-positive bacteria reveal walls that are thick, nearly uniformly dense layers (Figure 4.45A). In contrast, the cell walls of gram-negative bacteria are more complex, because in addition to a peptidoglycan layer they have another layer, called an outer membrane (Figure 4.45B). The structural differences between the cell walls of gram-positive and gram-negative bacteria reflect differences in biochemical composition. When techniques were developed to permit the separation of cell walls from cytoplasmic constituents, scientists could chemically analyze the cell wall. The major constituent found in the cell wall of gram-positive bacteria was peptidoglycan. It makes up about 40% to 80% of the dry weight of the wall, depending upon the species. Peptidoglycan has a tensile strength similar to that of reinforced concrete. Other constituents of gram-positive cell walls include teichoic acids and teichuronic acids. Teichoic acids are polyol phosphate polymers, such as polyglycerol phosphate and polyribitol phosphate (Figure 4.46). Sugars (such as glucose and galactose), amino sugars group of the first amino acid in the peptide cross-link is joined to the carboxyl group of the lactic acid moiety of N-acetylmuramic acid by a peptide bond. Some of the amino acids (e.g., alanine and glutamic acid) exist in the D stereoconfiguration. Note that one of the amino acids in the peptide—in Figure 4.42, lysine—is a diamino acid, that is, it contains two amino groups (Figure 4.43). It is essential that one of the amino acids in the cross-linking peptide bridge is a diamino acid, because the single amino groups of the other amino acids are tied up in peptide linkages all the way back to the muramic acid. The diamino acid can link one of its amino groups to one peptide chain and its other amino to the other chain, thus cross-linking (B) Gram-negative peptidoglycan (A) Gram-positive peptidoglycan the peptides and their amino sugar N-Acetylmuramic chains. The result of this cross-linkacid (NAM) ing is the formation of a twoN-Acetylglucosamine dimensional network around the (NAG) cell (Figure 4.44). It is this twodimensional sac that provides the rigidity and strength of the cell wall. Lysine and diaminopimelic acid (Figure 4.43) are the most comL-Alanine L-Alanine D-Glutamate D-Glutamate mon diamino acids responsible for L-Lysine L-DiaminoD-Alanine pimelic this cross-linking. Diaminopimelic acid (DAP) D -Alanine acid is not found in proteins; it Pentaglycine Direct cross-link cross-link occurs uniquely in the peptidoglycan of virtually all gram-negative Figure 4.44 Cell walls of gram-positive and gram-negative bacteria bacteria (Figure 4.44). The diagrams show the two-dimensional network of the peptidoglycan sac surConsiderable variation exists in Perry Staley Lory Microbiology 2/e rounding (A) a gram-positive and (B) a gram-negative cell. This layer is the major the amino acids that form the Sinauer Associates structural component of bacterial cell walls. The N-acetylglucosamine (NAG) and Ncross-linking peptides of peptidoacetylmuramic acid (NAM) are linked to form the amino sugar backbone (glycan). glycans. About 100 types of cell The glycan chains are held together by peptide bridges. STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA Milestones Box 4.4 The Gram Stain The Gram stain procedure was developed unwittingly in 1888 by Christian Gram, a Danish physician studying in Berlin. He was examining lung tissues during autopsies of individuals who had died of pneumonia. He noted that Streptococcus pneumoniae found in the lung tissues retained the primary stain known as Bismark Brown (crystal violet is now used),whereas the lung tissue did not.It was subsequently determined that certain bacteria, now termed gram-positive, like S.pneumoniae retain the purple dye when stained by this method, whereas others, the gram-negative bacteria, do not.The Gram stain is called a differential stain because it distinguishes between these two groups of bacteria. (A) 89 Procedure Color of the cells 1 Primary stain: prepare a smear of the bacterium and stain with crystal violet. 2 Mordant: flood the preparation with a solution of potassium iodide and iodine, which complexes with both the crystal violet and the cellular material. 3 Decolorization: add ethanol dropwise to the stained smear. 4 Counterstain: stain the preparation with safranin. Gram + Gram – Both gram-negative and gram-positive bacteria are stained purple. The dye complex is washed off the gramnegative bacteria. The gram-positive bacteria retain some of the dye and remain intensely purple in color. The gram-negative bacteria are now stained red. The gram-positive bacteria retain their purple color. The modern Gram stain procedure is illustrated here. Gram-positive bacteria have a single-layer, uniformly dense cell wall consisting primarily of peptidoglycan. Outside of cell Cell wall Cell membrane Periplasm Cytoplasm (B) Gram-negative bacteria have a two-layer cell wall consisting of a very thin peptidoglycan layer and an outer wall membrane. Outer membrane Cell wall Periplasm Peptidoglycan Cell membrane Cytoplasm Figure 4.45 Cell envelope structure Thin sections and diagrams showing the cell envelopes of (A) a gram-positive bacterium, Bacillus megaterium, and (B) the gram-negative bacterium Escherichia coli. Note the characteristic outer membrane of the gram-negative bacterium. The thin peptidoglycan layer of gram-negative bacteria is not always evident in electron micrographs. Note also the location of the periplasm in each type of organism. Photos courtesy of Peter Hirsch and Stuart Pankratz. 90 CHAPTER 4 (A) (B) (C) O– –O O– O –O O P P O –O O O CH2 CH2 P O O Glucose O CH D -Alanine O CH D -Alanine O CH H2C D -Alanine O O CH H2C O P O– D -Alanine –O O O– P CH CH2 O– O O Monomer CH2 –O O– P O O Figure 4.46 Teichoic acids Chemical structures of two teichoic acids found in gram-positive bacteria. Monomers of (A) the polyribitol teichoic acid of Bacillus subtilis and (B) the polyglycerol teichoic acid of Lactobacillus sp. (C) The monomers are joined by phosphate li