MCB3020 Textbook Chapter 3: Bacterial Cell Structure PDF

3 Bacterial Cell Structure ©Design Pics/Hammond HSN Hooking Up bacteria is explored, our understanding of them may change in interesting and exciting ways. E Readiness Check: ach year over 100 million people around the world become infected with Neisseria gonorrhoeae, the bacterium that causes...

3 Bacterial Cell Structure ©Design Pics/Hammond HSN Hooking Up bacteria is explored, our understanding of them may change in interesting and exciting ways. E Readiness Check: ach year over 100 million people around the world become infected with Neisseria gonorrhoeae, the bacterium that causes gonorrhea. This troubling statistic is made even more disturbing by the increasing resistance of the bacterium to the antibiotics used to treat the disease. In men, infection is usually readily detected, but for women, infection is often asymptomatic and can lead to serious consequences such as pelvic inflammatory disease (PID) and sterility. These concerns have led scientists to consider methods for preventing infection. One method is to block transmission. Unfortunately, relatively little is known about the transmission process except that it occurs during sexual intercourse and that numerous hairlike structures (called pili) covering the surface of the bacterium play a role in establishing infection. The bacterium uses pili for a type of movement called twitching motility and to adhere to surfaces such as the sperm and epithelial cells of its human host. It has long been thought that by attaching to sperm cells the bacterium could hitch a ride during sexual intercourse. This explained transmission between partners. However, it did not clarify how transmission from women to men occurs. It turns out that exposure of N. gonorrhoeae to seminal fluid increases its twitching motility and enhances formation of small clumps of bacteria. In addition, seminal fluid proteins appear to alter the morphology and function of pili. In particular these proteins cause bundles of pili to separate into single filaments, enhancing the interaction of bacterial cells with each other and with host surfaces. These changes evolved to promote infection of host epithelial cells and increase the likelihood of transmission during sexual intercourse from females to their male partner. As this story illustrates, even small, seemingly simple organisms such as bacteria can exhibit complex behaviors. To understand these amazing microbes, we must first examine their cell structure and begin to relate it to the functions they carry out. As we consider bacterial cell structure, it is important to remember that only about 1% of bacterial species have been cultured. Of the cultivated species, only a few have been studied in great detail. From this small sample, many generalizations are made, and it is presumed that most other bacteria are like the well-studied model organisms. However, part of the wonder and fun of science is that nature is full of surprises. As the biology of more and more Based on what you have learned previously, you should be able to: ✓ Describe the application of small subunit (SSU) rRNA analysis to the ✓ ✓ establishment of the three domain classification system proposed by Carl Woese (section 1.2) Identify the following structures or regions of a plant or animal cell and describe their functions: cell wall, plasma membrane, cytoplasm, mitochondria, chloroplasts, and ribosomes Define and give examples of essential nutrients; describe how they are used by cells 3.1 Use of the Term “Prokaryote” Is Controversial After reading this section, you should be able to: a. List the characteristics originally used to describe prokaryotic cells b. Debate the “prokaryote” controversy using current evidence about bacterial cells Bacteria and archaea have long been lumped together and referred to as prokaryotes. Although the term was first introduced early in the twentieth century, the concept of a prokaryote was not fully outlined until 1962, when R. Stanier and C. B. van Niel described prokaryotes in terms of what they lacked in comparison to eukaryotic cells. For instance, Stanier and van Niel pointed out that prokaryotes lack a membrane-bound nucleus, a cytoskeleton, membrane-bound organelles, and internal membranous structures such as the endoplasmic reticulum and Golgi apparatus. Since the 1960s, biochemical, genetic, and genomic analyses have shown that Bacteria and Archaea are distinct taxa. Because of these discoveries, Norman Pace proposed in 2006 that the term prokaryote should be abandoned and most microbiologists are in agreement. This controversy illustrates that microbiology is an exciting, dynamic, and rapidly changing field of study. Throughout this text, we avoid the term prokaryote and are as explicit as 40 wil11886_ch03_040-076.indd 40 22/10/18 7:12 pm 3.2 Bacteria Are Diverse but Share Some Common Features 41 possible about which characteristics are associated with members of Bacteria, which with members of Archaea, and which with members of both taxa. 3.2 Bacteria Are Diverse but Share Some Common Features After reading this section, you should be able to: a. Distinguish a typical bacterial cell from a typical plant or animal cell in terms of cell shapes and arrangements, size, and cell structures b. Discuss the factors that determine the size and shape of a bacterial cell Much of this chapter is devoted to a discussion of individual cell components. Therefore a preliminary overview of the features common to many bacterial cells is in order. We begin by considering overall cell morphology and then move to cell structures. Shape, Arrangement, and Size It might be expected that bacterial cells, being small and relatively simple, would be uniform in shape and size. This is not the case, as the microbial world offers considerable variety in terms of morphology. However, the two most common shapes are cocci and rods (figure 3.1). Cocci (s., coccus) are roughly spherical cells. They can exist singly or can be associated in characteristic arrangements that can be useful in their identification. Diplococci (s., diplococcus) arise when cocci divide and remain together to form pairs. Long chains of cocci result when cells adhere after repeated divisions in one plane; this pattern is seen in the genera Streptococcus, Enterococcus, and Lactococcus (figure 3.1a). Members of the (a) S. agalactiae—cocci in chains genus Staphylococcus divide in random planes to generate irregular, grapelike clusters (figure 3.1b). Divisions in two or three planes can produce symmetrical groupings of cocci. Bacteria in the genus Micrococcus often divide in two planes to form square groups of four cells called tetrads. In the genus Sarcina, cocci divide in three planes, producing cubical packets of eight cells. Legionella pneumophila is an example of a bacterium with a rod shape (figure 3.1c). Rods, sometimes called bacilli (s., bacillus), differ considerably in their length-to-width ratio, the coccobacilli being so short and wide that they resemble cocci. The shape of the rod’s end often varies between species and may be flat, rounded, football-shaped, or bifurcated. Although many rods occur singly, some remain together after division to form pairs or chains (e.g., Bacillus megaterium is found in long chains). There are several less common cell shapes and arrangements. Vibrios are comma-shaped (figure 3.2a). Spirilla are rigid, spiral-shaped cells (figure 3.2b). Many have tufts of flagella at one or both ends. Spirochetes are flexible, spiral-shaped bacteria that have a unique, internal flagellar arrangement (figure 3.2c). These bacteria are distinctive in other ways, and all belong to a single phylum, Spirochaetes. Some bacteria form stalks (e.g., Caulobacter crescentus) (figure 3.2d). Other bacteria are pleomorphic, being variable in shape and lacking a single, characteristic form. Phylum Spirochaetes (section 21.6); Caulobacteraceae and Hyphomicrobiaceae bacteria reproduce in unusual ways (section 22.1); Order Vibrionales includes aquatic bioluminescent bacteria and pathogens (section 22.3) Some bacteria can be thought of as multicellular. Many actinobacteria form long filaments called hyphae. The hyphae form a network called a mycelium (figure 3.2e), and in this sense, they are similar to eukaryotic filamentous fungi. Many cyanobacteria, a group of photosynthetic bacteria, are also filamentous. Being filamentous allows some degree of (b) S. aureus—cocci in clusters (c) L. pneumophila—rods in chains Figure 3.1 Cocci and Rods Are the Most Common Bacterial Shapes. These images are color-enhanced scanning electron micrographs. (a) Streptococcus agalactiae, the cause of group B streptococcal infections (×4,800). (b) Staphylococcus aureus. (c) Legionella pneumophila, the cause of Legionnaires’ disease. (a) ©Science Source; (b, c) Source: CDC/Janice Haney Carr. wil11886_ch03_040-076.indd 41 6/13/19 3:34 PM 42 CHAPTER 3 | Bacterial Cell Structure (a) V. vulnificus—comma-shaped vibrios (b) C. jejuni—spiral-shaped (c) L. interrogans—a spirochete (d) C. crescentus—a stalked bacterium (e) Streptomyces—a filamentous bacterium (f) C. crocatus fruiting body Figure 3.2 Other Cell Shapes and Aggregations. (a) Vibrio vulnificus, scanning electron micrograph (SEM, X13,184). (b) Campylobacter jejuni, SEM. (c) Leptospira interrogans, the spirochete that causes the disease leptospirosis. (d) Caulobacter crescentus, SEM. (e) Streptomyces sp., SEM. (f) Fruiting body of the myxobacterium Chondromyces crocatus. The fruiting body is composed of thousands of cells. (a) ©Media for Medical/Getty Images; (b) Source: Photo by DeWood, digital colorization by Stephen Ausmus/USDA-ARS; (c) ©Sebastian Kaulitzki/Getty Images; (d) ©Biology Pics/Science Source; (e) ©Dr. Amy Gehring; (f) ©Yoav Levy/DIOMEDIA differentiation among cells in the filament. For instance, some filamentous cyanobacteria form specialized cells within the filament, heterocysts, that carry out nitrogen fixation (see figure 21.10c). Myxobacteria are of particular note. These bacteria sometimes aggregate to form complex structures called fruiting bodies (figure 3.2f). Order Streptomycetales: an important source of antibiotics (section 23.1); Phylum Cyanobacteria: oxygenic photosynthetic bacteria (section 21.4); Order Myxococcales: bacteria with morphological complexity and multicellularity (section 22.4) Escherichia coli is an excellent representative of an averagesized bacterium. This rod-shaped bacterium is 1.1 to 1.5 μm wide by 2.0 to 6.0 μm long. However, the size range of bacterial cells extends far beyond this average (figure 3.3). Near the small end of the size continuum are members of the genus Mycoplasma (0.3 μm in diameter). At the other end of the continuum are bacteria such as some spirochetes, which can reach 500 μm in length, and the cyanobacterium Oscillatoria, which is about 7 μm in diameter (the same diameter as a red blood cell). Some bacteria are huge by “bacterial standards.” For instance, Epulopiscium fishelsoni grows as large as 600 by 80 μm, a little smaller than a printed hyphen and clearly larger than the well-known eukaryote Paramecium (figure 3.4). An even larger bacterium, Thiomargarita wil11886_ch03_040-076.indd 42 Specimen Red blood cell Approximate diameter or width × length in µm 7 E. coli 1.3 × 4.0 Streptococcus 0.8–1.0 Poxvirus 0.23 × 0.32 Influenza virus 0.085 T2 E. coli bacteriophage 0.065 × 0.095 Tobacco mosaic virus 0.015 × 0.300 Poliovirus 0.027 Figure 3.3 Sizes of Bacteria Relative to a Red Blood Cell and Viruses. The larger viruses are comparable in size to the smaller bacteria. 22/10/18 7:12 pm 3.2 Bacteria Are Diverse but Share Some Common Features 43 r r r = 1 µm Surface area = 12.6 µm2 Volume = 4.2 µm3 Surface =3 Volume r = 2 µm Surface area = 50.3 µm2 Volume = 33.5 µm3 Surface = 1.5 Volume Figure 3.5 The Surface-to-Volume Ratio Is an Important Determinant Figure 3.4 A Giant Bacterium. This phase-contrast micrograph shows Epulopiscium fishelsoni dwarfing the paramecia, which are protozoa. E. fishelsoni cells are about 530 µm long. of Cell Size. Surface area is calculated by the formula 4πr2. Volume is calculated by the formula 4/3πr3. Shape also affects the S/V ratio; rods with the same volume as a coccus have a greater S/V ratio. ©Esther Angert/Medical Images/DIOMEDIA namibiensis, lives in ocean sediment (see figure 22.20). Thus a few bacteria are much larger than the average eukaryotic cell (typical plant and animal cells are around 10 to 50 μm in diameter). The variety of sizes and shapes exhibited by bacteria raises a fundamental question: What causes a bacterial species to have a particular size and shape? Although far from being answered, recent discoveries have fueled a renewed interest in this question, and it is clear that size and shape determination are related and have been selected for during the evolutionary history of each bacterial species. For many years it was thought that microbes had to be small to increase the surface area-to-volume ratio (S/V ratio; figure 3.5). As this ratio increases, the uptake of nutrients and the diffusion of these and other molecules within the cell become more efficient, which in turn facilitates a rapid growth rate. Shape affects the S/V ratio. A rod with the same volume as a coccus has a higher S/V ratio than does the coccus. This means that a rod can have greater nutrient flux across its plasma membrane. However, the discovery of E. fishelsoni demonstrates that bacteria can be very large. For bacteria to be large, they must have other characteristics that maximize their S/V ratio, or their size must be beneficial in some way. For instance, E. fishelsoni has a highly convoluted plasma membrane, which increases its S/V ratio. In addition, large cells are less likely to be eaten by predatory protists. Cells that are filamentous, have stalks, or are oddly shaped are also less susceptible to predation. Cell Organization Structures often observed in bacterial cells are summarized and illustrated in table 3.1 and figure 3.6. Note that no single wil11886_ch03_040-076.indd 43 bacterium possesses all of these structures at all times. Some are found only in certain cells in certain conditions or in certain phases of the life cycle. There are several common features of bacterial cell structure. Bacterial cells are surrounded by several layers, which are collectively called the cell envelope. The most common cell envelope layers are the plasma membrane, cell wall, and capsule or slime layer. The innermost layer of the cell envelope is the plasma membrane, which surrounds the cytoplasm. Most bacteria have a chemically complex cell wall, which covers the plasma membrane. Many bacteria surround the cell wall with a capsule or slime layer. Because most bacteria do not contain internal, membrane-bound organelles, their interior appears morphologically simple. The genetic material is localized in a discrete region called the nucleoid and is not separated from the surrounding cytoplasm by membranes. Ribosomes and larger masses called inclusions are scattered about the cytoplasm. Finally, many bacteria use flagella for locomotion. In the remaining sections of this chapter, we describe these major structures observed in bacterial cells in more detail. Comprehension Check 1. Why is the term prokaryote considered an inadequate descriptor by some microbiologists? 2. What characteristic shapes can bacteria assume? Describe the ways in which bacterial cells cluster together. 3. What advantages might a bacterial species that forms multicellular arrangements (e.g., clusters or chains) have that are not afforded unicellular bacteria? 4. What is the relevance of the surface area-to-volume ratio? 22/10/18 7:12 pm 44 CHAPTER 3 Table 3.1 | Bacterial Cell Structure Common Bacterial Structures and Their Functions Capsule Ribosomes Cell wall Plasma membrane Nucleoid Fimbriae Chromosome (DNA) Inclusion Flagellum Figure 3.6 Structure of a Bacterial Cell. wil11886_ch03_040-076.indd 44 22/10/18 7:12 pm 3.3 Bacterial Plasma Membranes Control What Enters and Leaves the Cell 45 3.3 Bacterial Plasma Membranes Control What Enters and Leaves the Cell After reading this section, you should be able to: a. Describe the fluid mosaic model of membrane structure and identify the types of lipids typically found in bacterial membranes b. Distinguish macroelements (macronutrients) from trace elements (micronutrients) and provide examples of each c. Provide examples of growth factors needed by some microorganisms d. Compare and contrast passive diffusion, facilitated diffusion, active transport, and group translocation, and provide examples of each e. Discuss the challenge of iron uptake and describe how bacteria overcome this difficulty intracytoplasmic membrane systems. These internal membranes and the plasma membrane share a basic design. However, they can differ significantly in the lipids and proteins they contain. To understand these chemical differences and the many functions of the plasma membrane and other membranes, it is necessary to become familiar with membrane structure. Fluid Mosaic Model of Membrane Structure The most widely accepted model for membrane structure is the fluid mosaic model of Singer and Nicholson, which proposes that membranes are lipid bilayers within which proteins float (figure 3.7). Bacterial membranes have roughly equal amounts of lipids and proteins. Cell membranes are very thin structures, about 2 to 3 nm thick, that look like two dark lines on either side of a light interior when imaged by TEM. This characteristic appearance is evidence that the membrane is composed of two sheets of lipid molecules arranged end-to-end (figure 3.7). Cleavage of membranes by freeze-etching, a technique that reveals fine detail, exposes the proteins lying within the membrane lipid bilayer. Electron microscopes use beams of electrons to create highly magnified images (section 2.4); Scanning probe microscopy can visualize molecules and atoms (section 2.5) The chemical nature of membrane lipids is critical to their ability to form bilayers. Most membrane-associated lipids (e.g., the phospholipids shown in figure 3.7) are amphipathic: They are structurally asymmetric, with polar and nonpolar ends The cell envelope is defined as the plasma membrane and all the surrounding layers external to it. The cell envelopes of many bacteria consist of the plasma membrane, cell wall, and at least one additional layer (e.g., capsule or slime layer). Of all these layers, the plasma membrane is the most important because it encompasses the cytoplasm and defines the cell. If it is removed or compromised, the cell’s contents spill into the environment and the cell dies. Furthermore, despite being the innermost layer of the cell envelope, the plasma membrane is responsible for much of the cell’s relationship with the outside world. Thus we begin our consideration of bacterial cell structure by describing the plasma membrane. First, let’s consider what cells do to survive. Cells must interact in a selective fashion with their environment, acquire nutrients, and eliminate waste. They also have to maintain their interior in a constant, highly orIntegral Oligosaccharide ganized state in the face of external Glycolipid membrane changes. Plasma membranes are an protein Integral absolute requirement for all living ormembrane protein ganisms because they are involved in Hydrophobic carrying out these cellular tasks. amino acids A primary role of all plasma membranes is that they are selectively permeable barriers: They allow particular ions and molecules to pass either into or out of the cell, while preventing the movement of others. Thus the plasma membrane prevents the loss of essential components Phospholipid through leakage while allowing the Peripheral membrane movement of other molecules. Bacteprotein rial plasma membranes play additional critical roles. They are the location of Figure 3.7 The Fluid Mosaic Model of Bacterial Membrane Structure. This diagram shows the several crucial metabolic processes: integral membrane proteins (blue) floating in a lipid bilayer. Peripheral membrane proteins (purple) are respiration, photosynthesis, and the associated loosely with the inner membrane surface and/or integral membrane proteins. Small tan spheres synthesis of lipids and cell wall represent the hydrophilic ends of membrane phospholipids, and wiggly tails are the hydrophobic fatty constituents. acid chains. Phospholipids are drawn much larger than their actual size. Oligosaccharides (chains of In addition to the plasma memcarbohydrates) protrude into the environment and may be attached to proteins or to membrane brane, some bacteria have extensive phospholipids (glycolipids). wil11886_ch03_040-076.indd 45 22/10/18 7:12 pm 46 CHAPTER 3 | Bacterial Cell Structure NH+3 CH2 Ethanolamine CH2 Polar and hydrophilic end O –O P O O Glycerol CH2 CH CH2 O O C OC Bacterial Plasma Membranes Are Dynamic O CH2 CH2 CH2 CH2 Long, nonpolar, hydrophobic fatty acid chains CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 Fatty acids CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH3 Figure 3.8 The Structure of a Phospholipid. Phosphatidylethanolamine, a phospholipid often found in bacterial membranes. (figure 3.8). The polar ends interact with water and are hydrophilic; the nonpolar hydrophobic ends are insoluble in water and tend to associate with one another. In aqueous environments, amphipathic lipids can interact to form a bilayer. The outer surfaces of the bilayer are hydrophilic, whereas hydrophobic ends are buried in the interior away from the surrounding water (figure 3.7). Lipids (appendix I) Two types of membrane proteins have been identified based on their ability to be separated from the membrane. Peripheral membrane proteins are loosely connected to the membrane and can be easily removed (figure 3.7). They are soluble in aqueous solutions and make up about 20 to 30% of total membrane protein. The remaining proteins are integral membrane proteins. These are not easily extracted from membranes and are insoluble in aqueous solutions when freed of lipids. Integral membrane proteins, like membrane lipids, are amphipathic; their hydrophobic regions are buried in membrane lipids while the hydrophilic portions project from the membrane surface (figure 3.7). wil11886_ch03_040-076.indd 46 Integral membrane proteins carry out some of the most important functions of the membrane. Many are transport proteins used to move materials either into or out of the cell. Others are involved in energy-conserving processes, such as the proteins found in electron transport chains. Those integral membrane proteins with regions exposed to the outside of the cell enable the cell to interact with its environment. Proteins (appendix I) Bacterial membranes are lipid bilayers and many of their amphipathic lipids are phospholipids (figure 3.8). The plasma membrane is dynamic: The lipid composition varies with environmental temperature in such a way that the membrane remains fluid during growth. For example, bacteria growing at lower temperatures have more unsaturated fatty acids in their membrane phospholipids; that is, there are one or more double covalent bonds in the long hydrocarbon chains. At higher temperatures, their phospholipids have more saturated fatty acids—those in which the carbon atoms are connected only with single covalent bonds. Environmental factors affect microbial growth (section 7.5) Although most aspects of the fluid mosaic model are well supported by experimentation, the suggestion that membrane lipids and integral proteins are homogeneously distributed has been challenged by the discovery of microdomains. These regions of the membrane are formed by phospholipids in conjunction with other lipids like farnesol, hopanoids, and carotenoids (see figure 11.31). Hopanoids are similar in structure to cholesterol found in eukaryotic membranes (figure 3.9), and their rigid planar structure makes them more hydrophobic than phospholipids. The specific lipids found in microdomains vary among organisms, but they all regulate membrane rigidity and mark microdomain boundaries. HO (a) Cholesterol (a steroid) is found in the membranes of eukaryotes. OH OH OH OH (b) Bacteriohopanetetrol (a hopanoid) is found in many bacterial membranes. Figure 3.9 Membrane Steroids and Hopanoids. 22/10/18 7:12 pm 3.3 Bacterial Plasma Membranes Control What Enters and Leaves the Cell 47 Bacteria Use Many Mechanisms to Bring Nutrients into the Cell All plasma membranes function as barriers. Yet they must also allow movement of nutrients into the cell. If a microbe does not obtain nutrients from its environment, it will quickly exhaust its supply of amino acids, nucleotides, and other molecules needed to survive. In addition, if a microbe is to thrive and reproduce, it must have a source of energy. The energy source is used to generate the cell’s major energy currency: the high-energy molecule ATP. Clearly, obtaining energy and nutrient sources is one of the most important jobs an organism has, and it is primarily a function of the bacterial plasma membrane. Here we discuss nutrient uptake, but first let’s define some terms used to describe the nutrients needed by cells. Microbiologists refer to carbon, oxygen, hydrogen, nitrogen, sulfur, and phosphorus as macroelements or macronutrients because they are required in relatively large amounts. They are found in organic molecules such as proteins, lipids, nucleic acids, and carbohydrates. Other macroelements are potassium, calcium, magnesium, and iron. They exist as cations and generally are associated with and contribute to the activity and stability of molecules and cell structures such as enzymes and ribosomes. Thus they are important in many cellular processes, including protein synthesis and energy conservation. Enzymes and ribozymes speed up cellular chemical reactions (section 10.6); Translation in bacteria (section 13.7); Electron transport chains: sets of sequential redox reactions (section 10.4) Other elements are required in small amounts—amounts so small that in the lab they are often obtained as contaminants in water, glassware, and growth media. Likewise in nature, they are ubiquitous and usually present in adequate amounts to support the growth of microbes. Microbiologists call these elements micronutrients or trace elements. The micronutrients— manganese, zinc, cobalt, molybdenum, nickel, and copper—are needed by most cells. Micronutrients are part of certain enzymes, and they aid in catalysis of reactions and maintenance of protein structure. Some microbes are able to synthesize all the organic molecules they need from macroelements. However, some microbes are unable to synthesize certain molecules needed for survival. These molecules are called growth factors, and they must be obtained from the environment. There are three types of growth factors: amino acids, purines and pyrimidines, and vitamins. What are the common features of nutrient uptake by bacteria? Bacteria can only take in dissolved molecules. Uptake mechanisms are specific; that is, the necessary substances, and not others, are acquired. It does a cell no good to take in a substance wil11886_ch03_040-076.indd 47 that it cannot use. Bacteria are able to transport nutrients into the cell even when the concentration of a nutrient inside the cell is higher than the concentration outside. Thus they are able to move nutrients up a concentration gradient. This is important because bacteria often live in nutrient-poor habitats. In view of the enormous variety of nutrients and the complexity of the task, it is not surprising that bacteria use several different transport mechanisms: passive diffusion, facilitated diffusion, primary and secondary active transport, and group translocation. Passive Diffusion Passive diffusion, often called diffusion or simple diffusion, is the process by which molecules move from a region of higher concentration to one of lower concentration; that is, the molecules move down the concentration gradient. The rate of passive diffusion depends on the size of the concentration gradient between a cell’s exterior and its interior (figure 3.10). A large concentration gradient is required for adequate nutrient uptake by passive diffusion (i.e., the external nutrient concentration must be high while the internal concentration is low). The rate of diffusion decreases as more nutrient accumulates in the cell. This occurs if the nutrient is not used immediately upon entry. Most substances cannot freely diffuse into a cell. However, water and some gases, including O2 and CO2, easily cross the The plateau in this line represents the saturation effect that is seen whenever a carrier protein is involved in transport. Rate of transport The integral membrane proteins in microdomains differ from those in the bulk membrane, and are organized by proteins called flotillins. Flotillins are themselves integral membrane proteins, and they function to assemble large protein complexes like secretion systems for transporting molecules out of the cell and complexes that transmit signals from the environment to molecules in the cytoplasm. Carrier facilitated diffusion Passive diffusion Concentration gradient Figure 3.10 Passive and Facilitated Diffusion. The rate of diffusion depends on the size of the solute’s concentration gradient (the ratio of the extracellular concentration to the intracellular concentration). This example of facilitated diffusion involves a carrier protein that can be saturated. Sometimes facilitated diffusion is mediated by a channel. Channels often do not exhibit a saturation effect. 22/10/18 7:12 pm 48 CHAPTER 3 | Bacterial Cell Structure plasma membrane by passive diffusion. H2O also moves across membranes by passive diffusion. Larger molecules, ions, and polar substances must enter the cell by other mechanisms, all of which involve specialized proteins that are referred to as transport proteins. How Diffusion Works Facilitated Diffusion Outside cell Inside cell Outside cell Inside cell Carrier protein in its outward-facing conformation. It binds solute. After binding solute After releasing solute During facilitated diffusion, substances Carrier protein in move across the plasma membrane with the its inward-facing conformation. assistance of transport proteins that are either It releases solute channels or carriers. Channels, as their name into cell. indicates, are proteins that form pores in membranes through which substances can Figure 3.11 A Model of Facilitated Diffusion. Because there is no energy input, molecules pass; they are often involved in facilitated difcontinue to enter only as long as their concentration is greater on the outside. fusion. Channels show some specificity for the substances that pass through them, but this is considerably less than that shown by carriers, which are far more substrate specific. The rate of facilitated diffusion increases with the energy-dependent transport mechanisms capable of concentratconcentration gradient much more rapidly and at lower coning nutrients are significantly more important uptake mechacentrations of the diffusing molecule than that of passive difnisms for bacterial cells. fusion (figure 3.10). When the transporter is a carrier, the diffusion rate reaches a plateau above a specific gradient Primary and Secondary Active Transport value because the carrier protein is saturated; that is, it is transporting as many solute molecules as possible. The resultActive transport is the transport of solute molecules to ing curve resembles an enzyme-substrate curve (see figure 10.16) higher concentrations (i.e., against a concentration gradient) and is different from the linear response seen with passive with the input of metabolic energy. Three types of active diffusion. An example of channel-mediated facilitated diffutransport are observed in bacteria: primary active transport, sion is that involving aquaporins (see figure 2.31), which secondary active transport, and group translocation. They transport water. Aquaporins are members of the major differ in terms of the energy used to drive transport and intrinsic protein (MIP) family of proteins. MIPs facilitate difwhether or not the transported molecule is modified as it fusion of small polar molecules, and they are observed in all enters. organisms. Active transport resembles facilitated diffusion in that it Although facilitated diffusion relies on transport proteins, it involves carrier proteins. Recall that carrier proteins bind particular is truly diffusion because a concentration gradient spanning the solutes with great specificity. Active transport is also characterized membrane drives the movement of molecules, and no energy is by the carrier saturation effect at high solute concentrations used. If the concentration gradient disappears, net inward move(figure 3.10). Nevertheless, active transport differs from facilitated ment ceases. The gradient can be maintained by converting the diffusion because it uses metabolic energy and can concentrate transported nutrient to another compound, as occurs when a nusubstances within the cell. trient is metabolized. Primary active transport is mediated by carriers called Considerable work has been done on the mechanism of primary active transporters. They use energy provided by ATP carrier-mediated facilitated diffusion. When the solute molecule hydrolysis to move substances against a concentration gradient binds to the outside of the carrier, it changes conformation and without modifying them. Primary active transporters are unireleases the molecule on the cell interior (figure 3.11). The carporters; that is, they move a single molecule across the memrier subsequently changes back to its original shape and is ready brane (figure 3.12). ATP-binding cassette transporters (ABC to pick up another molecule. The net effect is that a hydrophilic transporters) are important primary active transporters. Our molecule can enter the cell in response to its concentration focus here is on those ABC transporters that are used for import gradient. of substances. Other ABC transporters are used for export Facilitated diffusion has been documented in some bacteria of substances, in particular proteins; these exporters are but it does not seem to be the major uptake mechanism described in chapter 13. Protein maturation and secretion for these microbes. Recall that many bacteria live in environ(section 13.8) ments where nutrient concentrations are low, and facilitated difMost ABC transporters consist of two hydrophobic fusion cannot concentrate nutrients inside cells. Therefore membrane-spanning regions (domains) with two ATP-binding wil11886_ch03_040-076.indd 48 22/10/18 7:12 pm 3.3 Bacterial Plasma Membranes Control What Enters and Leaves the Cell 49 Figure 3.13 ABC Transporter Function. Shown here is a transporter that The ion gradients used by secondary active transporters arise primarily in three ways. The first results from bacterial metabolic activity. During energy-conserving processes, electron transport generates a proton gradient in which protons are at a higher concentration outside the cell than inside. The proton gradient is used to do cellular work, including secondary active transport. Some bacteria use the second method, in which an enzyme called a V-type ATPase hydrolyzes ATP and uses the energy released to create either a proton gradient or a sodium gradient across the plasma membrane. Finally, a proton gradient can be used to create another ion gradient such as a sodium gradient. This is accomplished by an antiporter that brings protons in as sodium ions are moved out of the cell. The sodium gradient can then be used to drive uptake of nutrients by a symport mechanism. Electron transport and oxidative phosphorylation (step 3) generate the most ATP (section 11.6) The lactose permease of E. coli is a well-studied symport secondary active transporter. It is a single protein that transports a lactose molecule inward as a proton simultaneously enters the cell. The proton is moving down a proton gradient, and the energy released drives solute transport. X-ray diffraction studies show that the carrier protein exists in outward- and inwardfacing conformations. When lactose and a proton bind to separate sites on the outward-facing conformation, the protein changes to its inward-facing conformation, and the sugar and proton are released into the cytoplasm. Bacteria often have more than one transport system for a nutrient, as can be seen with E. coli. This bacterium has at least five transport systems for the sugar galactose, three systems each for the amino acids glutamate and leucine, and two potassium transport complexes. When several transport systems exist for the same substance, the systems differ in such properties as their energy source, their affinity for the solute transported, and the nature of their regulation. This diversity gives the bacterium an added competitive advantage in a variable environment. works with a solute-binding protein free in the periplasm. Other solutebinding proteins are associated with the plasma membrane, always associated with the transporter, or even fused to the transporter. Group Translocation Out In Uniporter Symporter Antiporter Cotransporters Figure 3.12 Carrier Proteins Can Be Uniporters or Cotransporters. Uniporters move a single substance into the cell. Cotransporters simultaneously move two substances across the membrane. When both substances move in the same direction, the carrier is a symporter. When the two substances move in opposite directions, the carrier is an antiporter. 1 After binding solute, the solute-binding protein approaches ABC transporter. 2 Solute-binding protein attaches to transporter and releases solute. Energy released by hydrolysis of ATP drives movement of solute Soluteacross membrane. binding protein Periplasm Transporter 1 2 Cytoplasm ATPbinding domain ATP ADP + Pi ATP ADP + Pi domains facing the cytoplasm (figure 3.13). The membranespanning domains form a pore in the membrane, and the ATPbinding domains bind and hydrolyze ATP to drive uptake. Most ABC transporters employ solute-binding proteins to deliver the molecule to be transported to the transporter. Secondary active transport couples the potential energy of ion gradients to transport of substances without modifying them. Secondary active transporters are cotransporters (figure 3.12). They move two substances simultaneously: the ion whose gradient powers transport and the substance being moved across the membrane. When the ion and other substance both move in the same direction, it is called symport. When they move in opposite directions, it is called antiport. Cotransport (Symport and Antiport) wil11886_ch03_040-076.indd 49 The distinguishing characteristic of group translocation is that a molecule is chemically modified as it is brought into the cell. The best-known group translocation system is the phosphoenolpyruvate: sugar phosphotransferase system (PTS), which is observed in many bacteria. The PTS transports a variety of sugars while phosphorylating them, using phosphoenolpyruvate (PEP) as the phosphate donor. PEP is a high-energy molecule that can be used to synthesize ATP, the cell’s energy currency. However, when it is used in PTS reactions, the energy present in PEP is used to energize sugar uptake rather than ATP synthesis. ATP: The major energy currency of cells (section 10.2) The transfer of phosphate from PEP to the incoming molecule involves several proteins and is an example of a phosphorelay system. In E. coli and Salmonella, the PTS consists of two enzymes and a low molecular weight heat-stable protein (HPr). A phosphate is transferred from PEP to enzyme II with the aid of 22/10/18 7:12 pm 50 CHAPTER 3 | Bacterial Cell Structure Phosphate is then transferred to incoming sugar via EIIB. Green - Fe3+ Red - O Gray - C Blue - N White - H ~ Mannitol-1-P P IIA IIB Pyruvate EI~ P HPr~ P HPr The high-energy phosphate of PEP is transferred via EI to HPr and from HPr to EIIA. IIC Mannitol Glucose-6-P P P ~ EI ~ PEP ~ P IIA IIB IIC Cytoplasm Glucose Periplasm Figure 3.14 Group Translocation: Bacterial PTS Transport. Two examples of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) are illustrated. The following components are involved in the system: phosphoenolpyruvate (PEP), enzyme I (EI), the low molecular weight heat-stable protein (HPr), and enzyme II (EII). EIIA is attached to EIIB in the mannitol transport system and is separate from EIIB in the glucose system. Figure 3.15 Enterobactin: A Siderophore Produced by E. coli. Ball-and-stick model of enterobactin complexed with Fe3+. Microorganisms secrete siderophores when iron is scarce in the medium. Once the iron-siderophore complex has reached the cell surface, it binds to a siderophore-receptor protein. Then either the iron is released to enter the cell directly or the whole iron-siderophore complex is transported inside by an ABC transporter. Iron is so crucial to microorganisms that more than one route of iron uptake may be used to ensure an adequate supply. Comprehension Check enzyme I and HPr (figure 3.14). Enzyme II then phosphorylates the sugar molecule as it is carried across the membrane. Many different PTSs exist, and they vary in terms of the sugars they transport. The specificity lies with the type of Enzyme II used in the PTS. Enzyme I and HPr are the same in all PTSs used by a bacterium. Enzymes and ribozymes speed up cellular chemical reactions (section 10.6) PTSs are widely distributed in bacteria, primarily among facultatively anaerobic bacteria (bacteria that grow in either the presence or absence of O2); some obligately anaerobic bacteria (e.g., Clostridium spp.) also have PTSs. However, most aerobic bacteria lack PTSs. Many carbohydrates are transported by PTSs. E. coli takes up glucose, fructose, mannitol, sucrose, N-acetylglucosamine, cellobiose, and other carbohydrates by group translocation. Active Transport by Group Translocation Iron Uptake Almost all microorganisms require iron for building molecules important in energy-conserving processes (e.g., cytochromes), as well as for the function of many enzymes. Iron uptake is made difficult by the extreme insolubility of ferric iron (Fe3+) and its derivatives, which leaves little free iron available for transport. Many bacteria overcome this difficulty by secreting siderophores (Greek for iron bearers). Siderophores are low molecular weight organic molecules that bind ferric iron and supply it to the cell (figure 3.15). Electron transport chains: sets of sequential redox reactions (section 10.4) wil11886_ch03_040-076.indd 50 1. List the functions of bacterial plasma membranes. Why must their plasma membranes carry out more functions than the plasma membranes of eukaryotic cells? 2. Describe in words and with a labeled diagram the fluid mosaic model for cell membranes. 3. On what basis are elements divided into macroelements and trace elements? 4. Describe facilitated diffusion, primary and secondary active transport, and group translocation in terms of their distinctive characteristics and mechanisms. What advantage does a bacterium gain by using active transport rather than facilitated diffusion? 5. What are uniport, symport, and antiport? 6. What are siderophores? Why are they important? 3.4 There Are Two Main Types of Bacterial Cell Walls After reading this section, you should be able to: a. Describe peptidoglycan structure b. Compare and contrast the cell walls of typical Gram-positive and Gram-negative bacteria c. Relate bacterial cell wall structure to the Gram-staining reaction The cell wall is the layer that lies just outside the plasma membrane. It is one of the most important structures for several reasons: 22/10/18 7:12 pm 3.4 There Are Two Main Types of Bacterial Cell Walls 51 It helps maintain cell shape and protect the cell from osmotic lysis; it can protect the cell from toxic substances; and in pathogens, it can contribute to pathogenicity. Cell walls are so important that most bacteria have them. Those that lack them have other features that fulfill cell wall function. Bacterial cell wall synthesis is an important target for many antibiotics. Antibacterial drugs (section 9.4) The typical Gram-positive cell envelope Peptidoglycan Plasma membrane Overview of Bacterial Cell Wall Structure After Christian Gram developed the Gram stain in 1884, it soon became evident that most bacteria could be divided into two major groups based on their response to the Gram-staining procedure. Gram-positive bacteria stained purple, whereas Gram-negative bacteria were pink or red. The true structural difference between these two groups did not become clear until the advent of the transmission electron microscope. Here we describe the long-held models of Gram-positive and Gram-negative cell walls developed from these studies. More recent studies of diverse groups of bacteria have shown that these models do not hold true for all bacteria (Microbial Diversity & Ecology 3.1). Because of ongoing discussions related to these new studies, we will refer to bacteria that fit the models as being typical Gram-positive or typical Gramnegative bacteria. Differential staining (section 2.3) The cell walls of Bacillus subtilis and many other typical Gram-positive bacteria consist of a single, 20- to 80-nm-thick homogeneous layer of peptidoglycan (murein) lying outside the plasma membrane (figure 3.16). In contrast, the cell walls of E. coli and many other typical Gram-negative bacteria have two distinct layers: a 2- to 7-nm-thick peptidoglycan layer covered by a 7- to 8-nm-thick outer membrane. One important feature seen in typical Gram-negative bacteria is a space between the plasma membrane and the outer membrane. It also is sometimes observed between the plasma membrane and cell wall in typical Gram-positive bacteria. This space is called the periplasmic space. The substance that occupies the periplasmic space is the periplasm. Cell wall The typical Gram-negative cell envelope Cell wall Outer membrane Peptidoglycan Plasma membrane Periplasmic space ©Egbert Hoiczyk Figure 3.16 Cell Envelopes of Typical Gram-Positive and Gram- Peptidoglycan Structure The feature common to nearly all bacterial cell walls is the presence of peptidoglycan, which forms an enormous meshlike structure often referred to as the peptidoglycan sacculus. Peptidoglycan is composed of many identical subunits. Each subunit within the sacculus contains two sugar derivatives, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), and several different amino acids. The amino acids form a short peptide, sometimes called the stem peptide, consisting of four alternating D- and Lamino acids; the peptide is connected to the carboxyl group of NAM (figure 3.17). Three of the amino acids are not found in proteins: D-glutamic acid, D-alanine, and meso-diaminopimelic acid. The presence of D-amino acids in the stem peptide protects against degradation by most peptidases, which recognize only the L-isomers of amino acid residues. The peptidoglycan subunit of many bacteria is shown in figure 3.17. Carbohydrates wil11886_ch03_040-076.indd 51 Negative Bacteria. Cell envelopes consist of the plasma membrane and any layers (e.g., cell wall) exterior to it. For simplicity, we show only the plasma membrane and cell wall. Staphylococcus aureus (top) has a typical Grampositive cell wall that consists primarily of peptidoglycan. Myxococcus xanthus (bottom) has a typical Gram-negative cell wall consisting of a thin layer of peptidoglycan, an outer membrane, and the periplasmic space. (appendix I); Proteins (appendix I); Proteins are polymers of amino acids (section 13.2) The peptidoglycan sacculus is formed by linking the sugars of the peptidoglycan subunits together to form a strand; the strands are then cross-linked to each other by covalent bonds formed between the stem peptides extending from each strand. As seen in figure 3.18, the backbone of each strand is composed of alternating NAG and NAM residues. The strand is helical, and the stem peptides extend out from the backbone in different directions. There are two types of cross-links: direct and indirect 22/10/18 7:12 pm 52 CHAPTER 3 | Bacterial Cell Structure MICROBIAL DIVERSITY & ECOLOGY 3.1 Gram Positive and Gram Negative or Monoderms and Diderms? The importance of the Gram stain in the history of microbiology cannot be overstated. The Gram stain reaction was for many years one of the critical pieces of information used by bacterial taxonomists to construct taxa, and it is still useful in identifying bacteria in clinical settings. The initial studies done to differentiate bacteria that stained Gram positive from those that stain Gram negative were done using model organisms such as Bacillus subtilis (Gram positive) and Escherichia coli (Gram negative). At the time, it was thought that all other bacteria would have similar cell wall structures. However, the long-held models of Gram-positive and Gram-negative cell walls do not hold true for all bacteria. Iain Sutcliffe has proposed that microbiologists stop referring to bacteria as either Gram positive or Gram negative. He suggests that instead we should more precisely describe bacterial cell envelope architectures by focusing on the observation that some bacteria have envelopes with a single membrane—the plasma membrane as seen in typical Gram-positive bacteria—while others have envelopes with two membranes—the plasma membrane and an outer membrane as seen in typical Gram-negative bacteria. He proposed calling the former monoderms and the latter diderms. But why make this change? Sutcliffe begins by pointing out that some bacteria staining Gram positive are actually diderms and some staining Gram negative are actually monoderms. By referring to Gram-positive-staining diderms as Gram-positive bacteria, it is too easy to mislead scientists and many a budding microbiologist into thinking that the bacterium via a peptide interbridge. A direct cross-link is characterized by connecting the carboxyl group of an amino acid in one stem peptide to the amino group of an amino acid in another stem peptide. For instance, many bacteria cross-link the strands by connecting the carboxyl group of the D-alanine at position 4 of the stem peptide directly to the amino group of diaminopimelic acid (position 3) of the other peptidoglycan strand’s stem peptide (the position 5 D-alanine is removed as the cross-link is formed). Bacteria that have indirect linkage use a peptide interbridge (also called an interpeptide bridge), a short chain of amino acids that links the stem peptide of one peptidoglycan strand to that of another (figure 3.19). Cross-linking results in one dense, interconnected network of peptidoglycan strands Synthesis of peptidoglycan occurs in the cytoplasm, at the plasma membrane, and in the periplasmic space (section 12.4) The peptidoglycan sacculus is strong but elastic. It is able to stretch and contract in response to osmotic pressure. This is due to the rigidity of the backbone coupled with the flexibility of the cross-links. Peptidoglycan sacculi are also rather porous, has a typical Gram-positive envelope. He also argues that by relating cell envelope architecture to the phylogenies of various bacterial taxa, we may gain insight into the evolution of these architectures. He notes that the phyla Firmicutes and Actinobacteria are composed almost completely of monoderm bacteria, whereas almost all other bacterial phyla consist of diderms. There are interesting exceptions to the relationship of phylogeny and cell envelope structure. For instance, members of the genus Mycobacterium (e.g., M. tuberculosis) belong to the predominantly monoderm phylum Actinobacteria. Mycobacteria have cell walls that consist of peptidoglycan and an outer membrane. The outer membrane is composed of mycolic acids rather than the phospholipids and lipopolysaccharides (LPSs) found in the typical Gram-negative cells’ outer membrane. Order Corynebacteriales includes important human pathogens (section 23.1) Members of the genus Deinococcus are another interesting exception. These bacteria stain Gram positive but are diderms. Their cell envelopes consist of the plasma membrane, what appears to be a typical Gram-negative cell wall, and an S-layer. Their outer membrane is distinctive because it lacks LPS. Deinococci are not unique in this respect, however. It is now known that members of several taxa have outer membranes that lack LPS. Source: Sutcliffe, I. C. 2010. A phylum level perspective on bacterial cell envelope architecture. Trends Microbiol. 18(10):464–70. allowing globular proteins having a molecular weight as large as 50,000 to pass through, depending on whether the sacculus is relaxed or stretched; thus only extremely large proteins are unable to pass through peptidoglycan. Variants of peptidoglycan are found, particularly among typical Gram-positive bacteria. For example, some substitute the diamino acid lysine for meso-diaminopimelic acid (figure 3.20) and cross-link chains via interpeptide bridges. These interpeptide bridges can vary considerably (figure 3.21). Peptidoglycan can also vary in terms of the length of the peptidoglycan strands and the amount of cross-linking. Bacteria that stain Gram positive tend to have much more cross-linking, whereas those that stain Gram negative have considerably less. Typical Gram-Positive Cell Walls Consist Primarily of Peptidoglycan Most cultured bacteria that stain Gram positive belong to the phyla Firmicutes and Actinobacteria, and most of these bacteria have 3.4 There Are Two Main Types of Bacterial Cell Walls 53 NAM NAG CH3 H NH C O CH2OH O H H O H OH H H H H O O H O H NH C O CH2OH O CH3 H3C CH C O NH CH3 C H C O L–Alanine NH H C Peptide side chain NAM NAG CH2 CH2 C O D–Glutamic acid Polysaccharide backbone COOH NH H C (CH2)3 CH C O NH2 COOH meso– Diaminopimelic acid NH H D–Alanine H D–Alanine C CH3 C O NH At this site, a bond may be formed to link this peptide to one on CH3 another chain. When this occurs, the terminal D-alanine is lost. O C C OH Figure 3.17 Peptidoglycan Subunit Composition. Shown is the peptidoglycan subunit of E. coli, many other typical Gram-negative bacteria, and many typical Gram-positive bacteria. This illustration shows the subunit before it has been inserted into the existing peptidoglycan polymer. NAG is N-acetylglucosamine. NAM is N-acetylmuramic acid. The stem peptide is composed of alternating D- and L-amino acids; it terminates with two D-alanines. The amino acids are shown in different colors for clarity. Figure 3.18 A Helical Peptidoglycan Strand. Because of the strand’s helical nature, the stem peptides project out in different directions from the NAM-NAG backbone. Here the stem peptides are shown projecting out at 90-degree angles. Some studies suggest that the angle is actually 120 degrees. NAM NAG L-Ala D-Glu D-Ala DAP DAP D-Ala D-Glu L-Ala NAM NAG (a) NAM NAG L-Ala D-GluNH2 D-Ala L-Lys thick cell walls composed of peptidoglycan and large amounts of other polymers such as teichoic acids (figure 3.22). Teichoic acids are polymers of glycerol or ribitol joined by phosphate groups (figure 3.23). Some teichoic acids are covalently linked to peptidoglycan and are referred to as wall teichoic acids. Others are covalently connected to the plasma membrane; they are called lipoteichoic acids. Wall teichoic acids extend beyond the surface of the peptidoglycan. They are negatively charged and help give the cell wall its negative charge. Teichoic acids are not present in other bacteria. wil11886_ch03_040-076.indd 53 D-Ala (b) Gly Gly Gly Peptide inte Gly rbridge Gly L-Lys D-GluNH2 L-Ala NAM NAG Figure 3.19 Peptidoglycan Cross-Links Can Be Direct or Indirect via a Peptide Interbridge. (a) E. coli peptidoglycan with direct cross-linking, typical of many Gram-negative bacteria. (b) Staphylococcus aureus peptidoglycan with an interbridge. S. aureus stains Gram positive. Gly is glycine. D-GluNH2 is D-glutamic acid with an NH2 group attached to the α carbon (the carbon next to the carboxyl group). 22/10/18 7:12 pm 54 | Bacterial Cell Structure CHAPTER 3 COOH H 2N C H 2N C H CH2 CH2 CH2 CH2 CH2 COOH H 2N NH2 (b) C C H CH2 CH2 H 2N CH2 (a) noncovalently bound to teichoic acids or other cell wall polymers, while others are covalently attached to the peptidoglycan. Membrane-bound enzymes called sortases catalyze the formation of covalent bonds that join these proteins to the peptidoglycan. Protein maturation and secretion (section 13.8) COOH H CH2 H Typical Gram-Negative Cell Walls Include Additional Layers Besides Peptidoglycan CH2 COOH (c) NH2 As just noted, most cultured bacteria that stain Gram positive belong to the phyla Firmicutes and Actinobacteria. With a few exceptions, bacteria belonging to the remaining phyla stain Gram negative (Microbial Diversity & Ecology 3.1). Even a brief inspection of figure 3.16 shows that typical Gram-negative cell walls are more complex than typical Gram-positive walls. One of the most striking differences is the paucity of peptidoglycan. The peptidoglycan layer is very thin (2 to 7 nm, depending on the bacterium) and sits within the periplasmic space. The periplasmic space is much larger than that of a typical Gram-positive cell, ranging from about 30 to 70 nm wide (figure 3.24). Some studies indicate that it may constitute about 20 to 40% of the total cell volume. The periplasmic space is home to a variety of proteins. Some periplasmic proteins participate in nutrient acquisition—for example, hydrolytic enzymes and transport proteins. Some periplasmic proteins are involved in energy conservation. For instance, some bacteria have electron transport proteins in their periplasm (e.g., denitrifying bacteria, which convert nitrate to nitrogen gas). Other periplasmic proteins are involved in peptidoglycan synthesis and modification of toxic compounds that could harm the cell. Anaerobic respiration uses the same three steps as aerobic respiration (section 11.7); Nitrogen cycle (section 28.1) The outer membrane lies outside the thin peptidoglycan layer. It is linked to the cell by Braun’s lipoprotein, the most abundant protein in the outer membrane (figure 3.24). This small Figure 3.20 Diamino Acids Present in Peptidoglycan. (a) L-lysine, (b) meso-diaminopimelic acid, (c) D -ornithine. Teichoic acids have several important functions. They help create and maintain the structure of the cell envelope by anchoring the wall to the plasma membrane. They are important during cell division, and they protect the cell from harmful substances in the environment (e.g., antibiotics and host defense molecules). In addition, they function in ion uptake and are involved in binding pathogenic species to host tissues, thus initiating the infectious disease process. The periplasmic space lies between the plasma membrane and the cell wall and is so narrow that it is often not visible by electron microscopy. The periplasm has relatively few proteins; this is probably because the peptidoglycan sacculus is so porous that many proteins translocated across the plasma membrane pass through the sacculus. Some secreted proteins are enzymes called exoenzymes. Exoenzymes often serve to degrade polymers such as proteins and polysaccharides that would otherwise be too large for transport across the plasma membrane; the degradation products, the monomer building blocks, are then taken up by the cell. Those proteins that remain in the periplasmic space are usually attached to the pla