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48 UNIT 1 Basic Principles of Microbiology together in groups or clusters after cell division, and the arrange- I Cell Shape and Size men...

48 UNIT 1 Basic Principles of Microbiology together in groups or clusters after cell division, and the arrange- I Cell Shape and Size ments are often characteristic of certain genera. For instance, some cocci form long chains (for example, the bacterium n this chapter we examine key structures of the prokaryotic I cell: the cytoplasmic membrane, the cell wall, cell surface structures and inclusions, and mechanisms of motility. Our over- Streptococcus), others occur in three-dimensional cubes (Sarcina), and still others in grapelike clusters (Staphylococcus). Several groups of bacteria are immediately recognizable by the arching theme will be structure and function. We begin this unusual shapes of their individual cells. Examples include spiro- chapter by considering two key features of prokaryotic cells— chetes, which are tightly coiled bacteria; appendaged bacteria, their shape and small size. Prokaryotes typically have defined which possess extensions of their cells as long tubes or stalks; and shapes and are extremely small cells. Shape is useful for differen- filamentous bacteria, which form long, thin cells or chains of cells tiating cells of the Bacteria and the Archaea and size has pro- (Figure 3.1). found effects on their biology. The cell morphologies shown here should be viewed with the understanding that they are representative shapes; many varia- 3.1 Cell Morphology tions of these key morphologies are known. For example, there In microbiology, the term morphology means cell shape. Several are fat rods, thin rods, short rods, and long rods, a rod simply morphologies are known among prokaryotes, and the most com- being a cell that is longer in one dimension than in the other. As mon ones are described by terms that are part of the essential we will see, there are even square bacteria and star-shaped bacte- lexicon of the microbiologist. ria! Cell morphologies thus form a continuum, with some shapes, such as rods, being very common and others more unusual. Major Cell Morphologies Examples of bacterial morphologies are shown in Figure 3.1. A Morphology and Biology bacterium that is spherical or ovoid in morphology is called a Although cell morphology is easily recognized, it is in general a coccus (plural, cocci). A bacterium with a cylindrical shape is poor predictor of other properties of a cell. For example, under called a rod or a bacillus. Some rods twist into spiral shapes and the microscope many rod-shaped Archaea look identical to rod- are called spirilla. The cells of many prokaryotic species remain shaped Bacteria, yet we know they are of different phylogenetic E. Canale-Parola Norbert Pfennig Coccus Spirochete Norbert Pfennig Norbert Pfennig Stalk Hypha Rod Budding and appendaged bacteria Norbert Pfennig T. D. Brock Spirillum Filamentous bacteria Figure 3.1 Representative cell morphologies of prokaryotes. Next to each drawing is a phase-contrast photomicrograph showing an example of that morphology. Organisms are coccus, Thiocapsa roseopersic- ina (diameter of a single cell = 1.5 ␮m); rod, Desulfuromonas acetoxidans (diameter = 1 ␮m); spirillum, Rhodospirillum rubrum (diameter = 1 ␮m); spirochete, Spirochaeta stenostrepta (diameter = 0.25 ␮m); budding and appendaged, Rhodomicrobium vannielii (diameter = 1.2 ␮m); filamentous, Chloroflexus aurantiacus (diameter = 0.8 ␮m).  CHAPTER 3 Cell Structure and Function in Bacteria and Archaea 49 domains ( Section 2.7). Thus, with very rare exceptions, it is UNIT 1 impossible to predict the physiology, ecology, phylogeny, or vir- tually any other property of a prokaryotic cell, by simply knowing its morphology. What sets the morphology of a particular species? Although we know something about how cell shape is controlled, we know little about why a particular cell evolved the morphology it has. Several selective forces are likely to be in play in setting the mor- phology of a given species. These include optimization for nutri- ent uptake (small cells and those with high surface-to-volume Esther R. Angert, Harvard University ratios), swimming motility in viscous environments or near sur- faces (helical or spiral-shaped cells), gliding motility (filamentous bacteria), and so on. Thus morphology is not a trivial feature of a microbial cell. A cell’s morphology is a genetically directed char- acteristic and has evolved to maximize fitness for the species in a particular habitat. (a) MiniQuiz How do cocci and rods differ in morphology? Is cell morphology a good predictor of other properties of the cell? 3.2 Cell Size and the Significance of Smallness Prokaryotes vary in size from cells as small as about 0.2 ␮m in diameter to those more than 700 ␮m in diameter (Table 3.1). The vast majority of rod-shaped prokaryotes that have been cultured in the laboratory are between 0.5 and 4 ␮m wide and less than 15 ␮m long, but a few very large prokaryotes, such as Epulopiscium Heidi Schulz fishelsoni, are huge, with cells longer than 600 ␮m (0.6 millimeter) (Figure 3.2). This bacterium, phylogenetically related to the endospore-forming bacterium Clostridium and found in the gut (b) of the surgeonfish, is interesting not only because it is so large, but also because it has an unusual form of cell division and contains Figure 3.2 Some very large prokaryotes. (a) Dark-field photomicro- graph of a giant prokaryote, Epulopiscium fishelsoni. The rod-shaped cell multiple copies of its genome. Multiple offspring are formed and in this field is about 600 ␮m (0.6 mm) long and 75 ␮m wide and is shown are then released from the Epulopiscium “mother cell.” A mother with four cells of the protist (eukaryote) Paramecium, each of which is cell of Epulopiscium contains several thousand genome copies, about 150 ␮m long. E. fishelsoni is a species of Bacteria, phylogenetically each of which is about the same size as the genome of Escherichia related to Clostridium. (b) Thiomargarita namibiensis, a large sulfur che- coli (4.6 million base pairs). The many copies are apparently nec- molithotroph (phylum Proteobacteria of the Bacteria) and currently the essary because the cell volume of Epulopiscium is so large (Table largest known prokaryote. Cell widths vary from 400 to 750 ␮m. 3.1) that a single copy of its genome would not be sufficient to support the transcriptional and translational needs of the cell. Cells of the largest known prokaryote, the sulfur che- dimensions of an average rod-shaped prokaryote, the bacterium molithotroph Thiomargarita (Figure 3.2b), can be 750 ␮m in E. coli, for example, are about 1 * 2 ␮m; these dimensions are diameter, nearly visible to the naked eye. Why these cells are so typical of most prokaryotes. For comparison, average eukaryotic large is not well understood, although for sulfur bacteria a large cells can be 10 to more than 200 ␮m in diameter. In general, then, cell size may be a mechanism for storing sulfur (an energy it can be said that prokaryotes are very small cells compared with source). It is hypothesized that problems with nutrient uptake eukaryotes. ultimately dictate the upper limits for the size of prokaryotic cells. Since the metabolic rate of a cell varies inversely with the Surface-to-Volume Ratios, Growth Rates, square of its size, for very large cells nutrient uptake eventually and Evolution limits metabolism to the point that the cell is no longer competi- There are significant advantages to being small. Small cells have tive with smaller cells. more surface area relative to cell volume than do large cells; that Very large cells are not common in the prokaryotic world. In is, they have a higher surface-to-volume ratio. Consider a spheri- contrast to Thiomargarita or Epulopiscium (Figure 3.2), the cal coccus. The volume of such a cell is a function of the cube of 50 UNIT 1 Basic Principles of Microbiology Table 3.1 Cell size and volume of some prokaryotic cells, from the largest to the smallest Organism Characteristics Morphology Sizea (μm) Cell volume (μm3) E. coli volumes Thiomargarita namibiensis Sulfur chemolithotroph Cocci in chains 750 200,000,000 100,000,000 a Epulopiscium fishelsoni Chemoorganotroph Rods with tapered ends 80 * 600 3,000,000 1,500,000 Beggiatoa speciesa Sulfur chemolithotroph Filaments 50 * 160 1,000,000 500,000 Achromatium oxaliferum Sulfur chemolithotroph Cocci 35 * 95 80,000 40,000 Lyngbya majuscula Cyanobacterium Filaments 8 * 80 40,000 20,000 Thiovulum majus Sulfur chemolithotroph Cocci 18 3,000 1500 Staphylothermus marinusa Hyperthermophile Cocci in irregular clusters 15 1,800 900 Magnetobacterium bavaricum Magnetotactic bacterium Rods 2 * 10 30 15 Escherichia coli Chemoorganotroph Rods 1*2 2 1 Pelagibacter ubiquea Marine chemoorganotroph Rods 0.2 * 0.5 0.014 0.007 Mycoplasma pneumoniae Pathogenic bacterium Pleomorphicb 0.2 0.005 0.0025 a Where only one number is given, this is the diameter of spherical cells. The values given are for the largest cell size observed in each species. For example, for T. namibiensis, an average cell is only about 200 ␮m in diameter. But on occasion, giant cells of 750 ␮m are observed. Likewise, an average cell of S. marinus is about 1 ␮m in diameter. The species of Beggiatoa here is unclear and E. fishel- soni and P. ubique are not formally recognized names in taxonomy. b Mycoplasma is a cell wall–less bacterium and can take on many shapes (pleomorphic means “many shapes”). Source: Data obtained from Schulz, H.N., and B.B. Jørgensen. 2001. Ann. Rev. Microbiol. 55: 105–137. its radius (V = 43 ␲r3), while its surface area is a function of the than larger cells, and a given amount of resources (the nutrients square of the radius (S = 4␲r2). Therefore, the S/V ratio of a available to support growth) will support a larger population of spherical coccus is 3/r (Figure 3.3). As a cell increases in size, its small cells than of large cells. How can this affect evolution? S/V ratio decreases. To illustrate this, consider the S/V ratio for Each time a cell divides, its chromosome replicates. As DNA is some of the cells of different sizes listed in Table 3.1: Pelagibacter replicated, occasional errors, called mutations, occur. Because ubique, 22; E. coli, 4.5; and E. fishelsoni, 0.05. mutation rates appear to be roughly the same in all cells, large or The S/V ratio of a cell affects several aspects of its biology, small, the more chromosome replications that occur, the greater including its evolution. For instance, because a cell’s growth rate the total number of mutations in the population. Mutations are depends, among other things, on the rate of nutrient exchange, the “raw material” of evolution; the larger the pool of mutations, the the higher S/V ratio of smaller cells supports a faster rate of greater the evolutionary possibilities. Thus, because prokaryotic nutrient exchange per unit of cell volume compared with that of cells are quite small and are also genetically haploid (allowing larger cells. Because of this, smaller cells, in general, grow faster mutations to be expressed immediately), they have, in general, the capacity for more rapid growth and evolution than larger, genetically diploid cells. In the latter, not only is the S/V ratio r = 1 ␮m smaller but the effects of a mutation in one gene can be masked r = 1 μm Surface area (4πr2 ) = 12.6 μm 2 by a second, unmutated gene copy. These fundamental differ- 4 Volume ( 3 πr3 ) = 4.2 μm 3 ences in size and genetics between prokaryotic and eukaryotic cells underlie the fact that prokaryotes can adapt quite rapidly to Surface changing environmental conditions and can more easily exploit =3 new habitats than can eukaryotic cells. We will see this concept Volume in action in later chapters when we consider, for example, the enormous metabolic diversity of prokaryotes, or the spread of r = 2 ␮m antibiotic resistance. r = 2 μm Surface area = 50.3 μm 2 Volume = 33.5 μm 3 Lower Limits of Cell Size From the foregoing discussion one might predict that smaller Surface and smaller bacteria would have greater and greater selective = 1.5 advantages in nature. However, this is not true, as there are lower Volume limits to cell size. If one considers the volume needed to house the essential components of a free-living cell—proteins, nucleic acids, ribosomes, and so on—a structure of 0.1 ␮m in diameter Figure 3.3 Surface area and volume relationships in cells. As a cell or less is simply insufficient to do the job, and structures 0.15 ␮m increases in size, its S/V ratio decreases.  CHAPTER 3 Cell Structure and Function in Bacteria and Archaea 51 in diameter are marginal. Thus, structures occasionally observed Glycerol UNIT 1 in nature of 0.1 ␮m or smaller that “look” like bacterial cells are O H almost certainly not so. Despite this, many very small prokary- C O C H otic cells are known and many have been grown in the laboratory. H3C O The open oceans, for example, contain 104–105 prokaryotic cells C O C H per milliliter, and these tend to be very small cells, 0.2–0.4 ␮m in H 3C O diameter. We will see later that many pathogenic bacteria are also Fatty acids H C O P O– very small. When the genomes of these pathogens are examined, H O they are found to be highly streamlined and missing many genes Phosphate CH2 whose functions are supplied to them by their hosts. CH2 Ethanolamine +NH (a) 3 MiniQuiz What physical property of cells increases as cells become smaller? Hydrophilic region How can the small size and haploid genetics of prokaryotes accelerate their evolution? Hydrophobic Fatty acids region II The Cytoplasmic Membrane Hydrophilic region and Transport (b) Glycerophosphates e now consider the structure and function of a critical cell W component, the cytoplasmic membrane. The cytoplasmic G. Wagner Fatty acids membrane plays many roles, chief among them as the “gate- keeper” for substances that enter and exit the cell. (c) 3.3 The Cytoplasmic Membrane Figure 3.4 Phospholipid bilayer membrane. (a) Structure of the phospholipid phosphatidylethanolamine. (b) General architecture of a The cytoplasmic membrane is a thin barrier that surrounds the bilayer membrane; the blue balls depict glycerol with phosphate and (or) cell and separates the cytoplasm from the cell’s environment. If other hydrophilic groups. (c) Transmission electron micrograph of a mem- the membrane is broken, the integrity of the cell is destroyed, the brane. The light inner area is the hydrophobic region of the model mem- cytoplasm leaks into the environment, and the cell dies. We will brane shown in part b. see that the cytoplasmic membrane confers little protection from osmotic lysis but is ideal as a selective permeability barrier. how extensive this is. The cytoplasmic membranes of some Composition of Membranes Bacteria are strengthened by molecules called hopanoids. These The general structure of the cytoplasmic membrane is a phos- somewhat rigid planar molecules are structural analogs of pholipid bilayer. Phospholipids contain both hydrophobic (fatty sterols, compounds that strengthen the membranes of eukaryotic acid) and hydrophilic (glycerol–phosphate) components and can cells, many of which lack a cell wall. be of many different chemical forms as a result of variation in the groups attached to the glycerol backbone (Figure 3.4) As phos- Membrane Proteins pholipids aggregate in an aqueous solution, they naturally form The major proteins of the cytoplasmic membrane have bilayer structures. In a phospholipid membrane, the fatty acids hydrophobic surfaces in their regions that span the membrane point inward toward each other to form a hydrophobic environ- and hydrophilic surfaces in their regions that contact the envi- ment, and the hydrophilic portions remain exposed to the exter- ronment and the cytoplasm (Figures 3.4 and 3.5). The outer sur- nal environment or the cytoplasm (Figure 3.4b). face of the cytoplasmic membrane faces the environment and in The cell’s cytoplasmic membrane, which is 6–8 nanometers gram-negative bacteria interacts with a variety of proteins that wide, can be seen with the electron microscope, where it appears bind substrates or process large molecules for transport into the as two dark-colored lines separated by a lighter area (Figure 3.4c). cell (periplasmic proteins, see Section 3.7). The inner side of the This unit membrane, as it is called (because each phospholipid cytoplasmic membrane faces the cytoplasm and interacts with leaf forms half of the “unit”), consists of a phospholipid bilayer proteins involved in energy-yielding reactions and other impor- with proteins embedded in it (Figure 3.5). Although in a diagram tant cellular functions. the cytoplasmic membrane may appear rather rigid, in reality it is Many membrane proteins are firmly embedded in the mem- somewhat fluid, having a consistency approximating that of a brane and are called integral membrane proteins. Other proteins low-viscosity oil. Some freedom of movement of proteins within have one portion anchored in the membrane and extramem- the membrane is possible, although it remains unclear exactly brane regions that point into or out of the cell (Figure 3.5). Still 52 UNIT 1 Basic Principles of Microbiology Out Phospholipids Hydrophilic groups 6–8 nm Hydrophobic groups In Integral membrane proteins Phospholipid molecule Figure 3.5 Structure of the cytoplasmic membrane. The inner surface (In) faces the cytoplasm and the outer surface (Out) faces the environment. Phospholipids compose the matrix of the cytoplasmic membrane with proteins embedded or surface associated. Although there are some chemical differences, the overall structure of the cytoplasmic membrane shown is similar in both prokaryotes and eukaryotes (but an excep- tion to the bilayer design is shown in Figure 3.7e). other proteins, called peripheral membrane proteins, are not point inward from each glycerol molecule are covalently linked. membrane-embedded but nevertheless remain firmly associated This forms a lipid monolayer instead of a lipid bilayer membrane with membrane surfaces. Some of these peripheral membrane (Figure 3.7d, e). In contrast to lipid bilayers, lipid monolayer proteins are lipoproteins, molecules that contain a lipid tail that membranes are extremely resistant to heat denaturation and are anchors the protein into the membrane. Peripheral membrane pro- therefore widely distributed in hyperthermophiles, prokaryotes teins typically interact with integral membrane proteins in impor- that grow best at temperatures above 808C. Membranes with a tant cellular processes such as energy metabolism and transport. mixture of bilayer and monolayer character are also possible, Proteins in the cytoplasmic membrane are arranged in clusters with some of the inwardly opposing hydrophobic groups cova- (Figure 3.5), a strategy that allows proteins that need to interact lently bonded while others are not. to be adjacent to one another. The overall protein content of the membrane is quite high, and it is thought that the variation in lipid bilayer thickness (6–8 nm) is necessary to accommodate O Ester Ether thicker and thinner patches of membrane proteins. H2C O C R O H2C O C R Archaeal Membranes HC O C R HC O C R In contrast to the lipids of Bacteria and Eukarya in which ester O O linkages bond the fatty acids to glycerol, the lipids of Archaea CH3 H2C O P O– H 2C O P O– contain ether bonds between glycerol and their hydrophobic side H2C C C CH2 chains (Figure 3.6). Archaeal lipids lack true fatty acid side chains O– O– H and instead, the side chains are composed of repeating units of Bacteria Archaea the hydrophobic five-carbon hydrocarbon isoprene (Figure 3.6c). Eukarya The cytoplasmic membrane of Archaea can be constructed of (a) (b) (c) either glycerol diethers (Figure 3.7a), which have 20-carbon side Figure 3.6 General structure of lipids. (a) The ester linkage and chains (the 20-C unit is called a phytanyl group), or diglycerol (b) the ether linkage. (c) Isoprene, the parent structure of the hydropho- tetraethers (Figure 3.7b), which have 40-carbon side chains. In bic side chains of archaeal lipids. By contrast, in lipids of Bacteria and the tetraether lipid, the ends of the phytanyl side chains that Eukarya, the side chains are composed of fatty acids (see Figure 3.4a).  CHAPTER 3 Cell Structure and Function in Bacteria and Archaea 53 Phytanyl UNIT 1 CH3 H2C O C CH3 HC O C H2COPO32– CH3 groups (a) Glycerol diether Isoprene unit Biphytanyl –2 OPOCH H2C O C 3 2 C O CH HC O C H2COPO32– C O CH2 (b) Diglycerol tetraethers HOH2C HC O C H 2C O C C O CH2 (c) Crenarchaeol C O CH CH2OH Out Out Glycerophosphates Phytanyl Biphytanyl Membrane protein In In (d) Lipid bilayer (e) Lipid monolayer Figure 3.7 Major lipids of Archaea and the architecture of archaeal membranes. (a, b) Note that the hydrocarbon of the lipid is attached to the glycerol by an ether linkage in both cases. The hydrocarbon is phytanyl (C20) in part a and biphytanyl (C40) in part b. (c) A major lipid of Crenarchaeota is crenarchaeol, a lipid containing 5- and 6-carbon rings. (d, e) Membrane structure in Archaea may be bilayer or monolayer (or a mix of both). Many archaeal lipids also contain rings within the hydrocar- overall membrane function), and considerable variation in the bon chains. For example, crenarchaeol, a lipid widespread among number and position of the rings has been discovered in the species of Crenarchaeota ( Section 2.10), contains four lipids of different species. cyclopentyl rings and one cyclohexyl ring (Figure 3.7c). The pre- Despite the differences in chemistry between the cytoplasmic dominant membrane lipids of many Euryarchaeota, such as the membranes of Archaea and organisms in the other domains, methanogens and extreme halophiles, are glycolipids, lipids with the fundamental construction of the archaeal cytoplasmic a carbohydrate bonded to glycerol. Rings formed in the hydro- membrane—inner and outer hydrophilic surfaces and a hydropho- carbon side chains affect the properties of the lipids (and thus bic interior—is the same as that of membranes in Bacteria and 54 UNIT 1 Basic Principles of Microbiology Eukarya. Evolution has selected this design as the best solution to the main function of the cytoplasmic membrane—permeability— Table 3.2 Comparative permeability of membranes to various molecules and we consider this problem now. Potential for diffusion MiniQuiz Substance Rate of permeabilitya into a cell Draw the basic structure of a lipid bilayer and label the Water 100 Excellent hydrophilic and hydrophobic regions. Glycerol 0.1 Good How are the membrane lipids of Bacteria and Archaea similar, Tryptophan 0.001 Fair/Poor and how do they differ? Glucose 0.001 Fair/Poor Chloride ion (Cl2) 0.000001 Very poor 3.4 Functions of the Cytoplasmic Potassium ion (K1) 0.0000001 Extremely poor Membrane 1 Sodium ion (Na ) 0.00000001 Extremely poor The cytoplasmic membrane is more than just a barrier separat- a Relative scale—permeability with respect to permeability to water given as 100. Perme- ing the inside from the outside of the cell. The membrane plays ability of the membrane to water may be affected by aquaporins (see text). critical roles in cell function. First and foremost, the membrane functions as a permeability barrier, preventing the passive leak- age of solutes into or out of the cell (Figure 3.8). Secondly, the One substance that does freely pass the membrane in both membrane is an anchor for many proteins. Some of these are directions is water, a molecule that is weakly polar but suffi- enzymes that catalyze bioenergetic reactions and others trans- ciently small to pass between phospholipid molecules in the lipid port solutes into and out of the cell. We will learn in the next bilayer (Table 3.2). But in addition, the movement of water across chapter that the cytoplasmic membrane is also a major site of the membrane is accelerated by dedicated transport proteins energy conservation in the cell. The membrane has an energeti- called aquaporins. For example, aquaporin AqpZ of Escherichia cally charged form in which protons (H1) are separated from coli imports or exports water depending on whether osmotic hydroxyl ions (OH2) across its surface (Figure 3.8). This charge conditions in the cytoplasm are high or low, respectively. The rel- separation is a form of energy, analogous to the potential energy ative permeability of the membrane to a few biologically relevant present in a charged battery. This energy source, called the substances is shown in Table 3.2. As can be seen, most sub- proton motive force, is responsible for driving many energy- stances cannot diffuse into the cell and thus must be transported. requiring functions in the cell, including some forms of trans- port, motility, and biosynthesis of ATP. Transport Proteins Transport proteins do more than just ferry substances across the The Cytoplasmic Membrane membrane—they accumulate solutes against the concentration as a Permeability Barrier gradient. The necessity for carrier-mediated transport is easy to The cytoplasm is a solution of salts, sugars, amino acids, understand. If diffusion were the only mechanism by which nucleotides, and many other substances. The hydrophobic por- solutes entered a cell, cells would never achieve the intracellular tion of the cytoplasmic membrane (Figure 3.5) is a tight barrier concentrations necessary to carry out biochemical reactions; that to diffusion of these substances. Although some small hydropho- is, their rate of uptake and intracellular concentration would bic molecules pass the cytoplasmic membrane by diffusion, polar never exceed the external concentration, which in nature is often and charged molecules do not diffuse but instead must be trans- quite low (Figure 3.9). Hence, cells must have mechanisms for ported. Even a substance as small as a proton (H1) cannot diffuse accumulating solutes—most of which are vital nutrients—to levels across the membrane. higher than those in their habitats, and this is the job of transport proteins. + ++ + + + + + + + + + + + + + + + + + + – – – – – – – – – – – – – – – – –– + + –– – + + – – – + – + – OH - – + + – – – – – – – – – – – – – – + ++ + ++ + + + + + + + + + + + + + + + + H (a) Permeability barrier: (b) Protein anchor: (c) Energy conservation: Prevents leakage and functions as a Site of many proteins that participate in Site of generation and use of the gateway for transport of nutrients into, transport, bioenergetics, and chemotaxis proton motive force and wastes out of, the cell Figure 3.8 The major functions of the cytoplasmic membrane. Although structurally weak, the cytoplasmic membrane has many important cellular functions.  CHAPTER 3 Cell Structure and Function in Bacteria and Archaea 55 Simple transport: Out In UNIT 1 Driven by the energy in the proton motive H+ force Transporter saturated H+ Rate of solute entry with substrate Transported Transport substance Group translocation: P Chemical modification Simple diffusion of the transported R~P substance driven by phosphoenolpyruvate External concentration of solute 1 Figure 3.9 Transport versus diffusion. In transport, the uptake rate 2 shows saturation at relatively low external concentrations. ABC transporter: 3 Periplasmic binding ATP ADP + Pi Transport systems show several characteristic properties. proteins are involved First, in contrast with diffusion, transport systems show a and energy comes from ATP saturation effect. If the concentration of substrate is high enough to saturate the transporter, which can occur at even the very low Figure 3.10 The three classes of transport systems. Note how substrate concentrations found in nature, the rate of uptake simple transporters and the ABC system transport substances without becomes maximal and the addition of more substrate does not chemical modification, whereas group translocation results in chemical increase the rate (Figure 3.9). This characteristic feature of trans- modification (in this case phosphorylation) of the transported substance. port proteins is essential for a system that must concentrate The three proteins of the ABC system are labeled 1, 2, and 3. nutrients from an often very dilute environment. A second char- acteristic of carrier-mediated transport is the high specificity of the transport event. Many carrier proteins react only with a sin- Structure and Function of Membrane gle molecule, whereas a few show affinities for a closely related Transport Proteins class of molecules, such as sugars or amino acids. This economy At least three transport systems exist in prokaryotes: simple in uptake reduces the need for separate transport proteins for transport, group translocation, and ABC transport. Simple trans- each different amino acid or sugar. port consists only of a membrane-spanning transport protein, And finally, a third major characteristic of transport systems is group translocation involves a series of proteins in the transport that their biosynthesis is typically highly regulated by the cell. event, and the ABC system consists of three components: a That is, the specific complement of transporters present in the substrate-binding protein, a membrane-integrated transporter, cytoplasmic membrane of a cell at any one time is a function of and an ATP-hydrolyzing protein (Figure 3.10). All transport sys- both the resources available and their concentrations. Biosyn- tems require energy in some form, either from the proton motive thetic control of this type is important because a particular nutri- force, or ATP, or some other energy-rich organic compound. ent may need to be transported by one type of transporter when Figure 3.10 contrasts these transport systems. Regardless of the nutrient is present at high concentration and by a different, the system, the membrane-spanning proteins typically show sig- higher-affinity transporter, when present at low concentration. nificant similarities in amino acid sequence, an indication of the common evolutionary roots of these structures. Membrane MiniQuiz transporters are composed of 12 alpha helices that weave back List two reasons why a cell cannot depend on diffusion as a and forth through the membrane to form a channel. It is through means of acquiring nutrients. this channel that a solute is actually carried into the cell (Figure Why is physical damage to the cytoplasmic membrane such a 3.11). The transport event requires that a conformational change critical issue for the cell? occur in the membrane protein following binding of its solute. Like a gate swinging open, the conformational change then brings the solute into the cell. 3.5 Transport and Transport Systems Actual transport events can be of three types: uniport, symport, and antiport (Figure 3.11). Uniporters are proteins that transport a Nutrient transport is a vital process. To fuel metabolism and sup- molecule unidirectionally across the membrane, either in or out. port growth, cells need to import nutrients and export wastes on Symporters are cotransporters; they transport one molecule along a continuous basis. To fulfill these requirements, several different with another substance, typically a proton. Antiporters are pro- mechanisms for transport exist in prokaryotes, each with its own teins that transport one molecule into the cell while simultane- unique features, and we explore this subject here. ously transporting a second molecule out of the cell. 56 UNIT 1 Basic Principles of Microbiology activity is the energy-driven accumulation of lactose in the cyto- plasm against the concentration gradient. Out Group Translocation: The Phosphotransferase System Group translocation is a form of transport in which the sub- stance transported is chemically modified during its uptake across the membrane. One of the best-studied group translocation sys- tems transports the sugars glucose, mannose, and fructose in E. coli. These compounds are modified by phosphorylation during In transport by the phosphotransferase system. The phosphotransferase system consists of a family of proteins Uniporter Antiporter Symporter that work in concert; five proteins are necessary to transport any given sugar. Before the sugar is transported, the proteins in the Figure 3.11 Structure of membrane-spanning transporters and phosphotransferase system are themselves alternately phosphor- types of transport events. Membrane-spanning transporters are made ylated and dephosphorylated in a cascading fashion until the of 12 α-helices (each shown here as a cylinder) that aggregate to form a channel through the membrane. Shown here are three different transport actual transporter, Enzyme IIc, phosphorylates the sugar during events; for antiporters and symporters, the cotransported substance is the transport event (Figure 3.13). A small protein called HPr, the shown in yellow. enzyme that phosphorylates HPr (Enzyme I), and Enzyme IIa are all cytoplasmic proteins. By contrast, Enzyme IIb lies on the inner surface of the membrane and Enzyme IIc is an integral mem- Simple Transport: Lac Permease of Escherichia coli brane protein. HPr and Enzyme I are nonspecific components of The bacterium Escherichia coli metabolizes the disaccharide the phosphotransferase system and participate in the uptake of sugar lactose. Lactose is transported into cells of E. coli by the several different sugars. Several different versions of Enzyme II activity of a simple transporter, lac permease, a type of sym- exist, one for each different sugar transported (Figure 3.13). porter. This is shown in Figure 3.12, where the activity of lac per- Energy for the phosphotransferase system comes from the mease is compared with that of some other simple transporters, energy-rich compound phosphoenolpyruvate, which is a key including uniporters and antiporters. We will see later that lac intermediate in glycolysis, a major pathway for glucose metabo- permease is one of three proteins required to metabolize lactose lism present in most cells ( Section 4.8). in E. coli and that the synthesis of these proteins is highly regu- lated by the cell ( Section 8.5). Periplasmic Binding Proteins and the ABC System As is true of all transport systems, the activity of lac permease We will learn a bit later in this chapter that gram-negative bacte- is energy-driven. As each lactose molecule is transported into the ria contain a region called the periplasm that lies between the cell, the energy in the proton motive force (Figure 3.8c) is dimin- cytoplasmic membrane and a second membrane layer called the ished by the cotransport of protons into the cytoplasm. The outer membrane, part of the gram-negative cell wall (Section 3.7). membrane is reenergized through energy-yielding reactions that The periplasm contains many different proteins, several of which we will describe in Chapter 4. Thus the net result of lac permease function in transport and are called periplasmic binding proteins. H+ K+ H+ H+ HSO4– HPO42– Na+ Lactose Out In Sulfate Potassium Phosphate H+ Sodium–proton Lac permease symporter uniporter symporter antiporter (a symporter) Figure 3.12 The lac permease of Escherichia coli and several other well-characterized simple transporters. Note the different classes of transport events depicted.  CHAPTER 3 Cell Structure and Function in Bacteria and Archaea 57 Glucose UNIT 1 Out Cytoplasmic membrane Nonspecific components Specific components Enz IIc Direction of glucose PE P transport Enz HPr Enz Enz I IIa IIb Pyruvate P P In Direction of P transfer P Glucose 6_P Figure 3.13 Mechanism of the phosphotransferase system of Escherichia coli. For glucose uptake, the system consists of five proteins: Enzyme (Enz) I, Enzymes IIa, IIb, and IIc, and HPr. A phosphate cascade occurs from phosphoenolpyruvate (PE-P) to Enzyme IIc and the latter actually transports and phosphory- lates the sugar. Proteins HPr and Enz I are nonspecific and transport any sugar. The Enz II components are specific for each particular sugar. Transport systems that employ periplasmic binding proteins along because they are tagged in a specific way. We discuss this process with a membrane transporter and ATP-hydrolyzing proteins are later ( Section 6.21). called ABC transport systems, the “ABC” standing for ATP- Protein export is important to bacteria because many bacterial binding cassette, a structural feature of proteins that bind ATP enzymes are designed to function outside the cell (exoenzymes). (Figure 3.14). More than 200 different ABC transport systems have For example, hydrolytic exoenzymes such as amylase or cellulase been identified in prokaryotes. ABC transporters exist for the are excreted directly into the environment where they cleave uptake of organic compounds such as sugars and amino acids, inor- starch or cellulose, respectively, into glucose; the glucose is then ganic nutrients such as sulfate and phosphate, and trace metals. used by the cell as a carbon and energy source. In gram-negative A characteristic property of periplasmic binding proteins is their high substrate affinity. These proteins can bind their sub- strate(s) even when they are at extremely low concentration; for example, less than 1 micromolar (1026 M). Once its substrate is Peptidoglycan bound, the periplasmic binding protein interacts with its respec- tive membrane transporter to transport the substrate into the cell Periplasmic Periplasm binding protein driven by ATP hydrolysis (Figure 3.14). Even though gram-positive bacteria lack a periplasm, they Transported have ABC transport systems. In gram-positive bacteria, however, substance Out substrate-binding proteins are anchored to the external surface of the cytoplasmic membrane. Nevertheless, once these proteins bind substrate, they interact with a membrane transporter to cat- alyze uptake of the substrate at the expense of ATP hydrolysis, just as they do in gram-negative bacteria (Figure 3.14). Membrane- spanning Protein Export transporter Thus far our discussion of transport has focused on small mole- cules. How do large molecules, such as proteins, get out of cells? ATP- Many proteins need to be either transported outside the cyto- hydrolyzing plasmic membrane or inserted in a specific way into the mem- In protein brane in order to function properly. Proteins are exported through and inserted into prokaryotic membranes by the activi- 2 ATP 2 ADP + 2 Pi ties of other proteins called translocases, a key one being the Sec (sec for secretory) system. The Sec system both exports proteins Figure 3.14 Mechanism of an ABC transporter. The periplasmic binding protein has high affinity for substrate, the membrane-spanning and inserts integral membrane proteins into the membrane. Pro- proteins form the transport channel, and the cytoplasmic ATP-hydrolyzing teins destined for transport are recognized by the Sec system proteins supply the energy for the transport event. 58 UNIT 1 Basic Principles of Microbiology bacteria, many enzymes are periplasmic enzymes, and these called peptidoglycan, is a polysaccharide composed of two sugar must traverse the cytoplasmic membrane in order to function. derivatives—N-acetylglucosamine and N-acetylmuramic acid— Moreover, many pathogenic bacteria excrete protein toxins or and a few amino acids, including L-alanine, D-alanine, D-glutamic other harmful proteins into the host during infection. Many tox- acid, and either lysine or the structurally similar amino acid analog, ins are excreted by a second translocase system called the type III diaminopimelic acid (DAP). These constituents are connected to secretion system. This system differs from the Sec system in that form a repeating structure, the glycan tetrapeptide (Figure 3.16). the secreted protein is translocated from the bacterial cell Long chains of peptidoglycan are biosynthesized adjacent to directly into the host, for example, a human cell. However, all of one another to form a sheet surrounding the cell (see Figure these large molecules need to move through the cytoplasmic 3.18). The chains are connected through cross-links of amino membrane, and translocases such as SecYEG and the type III acids. The glycosidic bonds connecting the sugars in the glycan secretion system assist in these transport events. strands are covalent bonds, but these provide rigidity to the structure in only one direction. Only after cross-linking is pepti- MiniQuiz doglycan strong in both the X and Y directions (Figure 3.17). Contrast simple transporters, the phosphotransferase system, Cross-linking occurs to different extents in different species of and ABC transporters in terms of (1) energy source, (2) chemical Bacteria; more extensive cross-linking results in greater rigidity. alterations of the solute transported, and (3) number of proteins In gram-negative bacteria, peptidoglycan cross-linkage occurs involved. by peptide bond formation from the amino group of DAP of Which transport system is best suited for the transport of nutri- one glycan chain to the carboxyl group of the terminal D-alanine ents present at extremely low levels, and why? on the adjacent glycan chain (Figure 3.17). In gram-positive bac- Why is protein excretion important to cells? teria, cross-linkage may occur through a short peptide inter- bridge, the kinds and numbers of amino acids in the interbridge varying from species to species. For example, in the gram-positive III Cell Walls of Prokaryotes Staphylococcus aureus, the interbridge peptide is composed of five glycine residues, a common interbridge amino acid (Figure 3.17b). The overall structure of peptidoglycan is shown in 3.6 The Cell Wall of Bacteria: Figure 3.17c. Peptidoglycan Peptidoglycan can be destroyed by certain agents. One such agent is the enzyme lysozyme, a protein that cleaves the Because of the activities of transport systems, the cytoplasm of β-1,4-glycosidic bonds between N-acetylglucosamine and bacterial cells maintains a high concentration of dissolved N-acetylmuramic acid in peptidoglycan (Figure 3.16), thereby solutes. This causes a significant osmotic pressure—about 2 weakening the wall; water can then enter the cell and cause lysis. atmospheres in a typical bacterial cell. This is roughly the same as Lysozyme is found in animal secretions including tears, saliva, the pressure in an automobile tire. To withstand these pressures and other body fluids, and functions as a major line of defense and prevent bursting (cell lysis), bacteria employ cell walls. against bacterial infection. When we consider peptidoglycan Besides protecting against osmotic lysis, cell walls also confer biosynthesis in Chapter 5 we will see that the important antibi- shape and rigidity on the cell. otic penicillin also targets peptidoglycan, but in a different way Species of Bacteria can be divided into two major groups, from that of lysozyme. Whereas lysozyme destroys preexisting called gram-positive and gram-negative. The distinction peptidoglycan, penicillin instead prevents its biosynthesis, lead- between gram-positive and gram-negative bacteria is based on ing eventually to osmotic lysis. the Gram stain reaction ( Section 2.2). But differences in cell wall structure are at the heart of the Gram stain reaction. The surface of gram-positive and gram-negative cells as viewed in the Diversity of Peptidoglycan Peptidoglycan is present only in species of Bacteria—the sugar electron microscope differs markedly, as shown in Figure 3.15. N-acetylmuramic acid and the amino acid analog DAP have The gram-negative cell wall, or cell envelope as it is sometimes never been found in the cell walls of Archaea or Eukarya. How- called, is chemically complex and consists of at least two layers, ever, not all Bacteria examined have DAP in their peptidoglycan; whereas the gram-positive cell wall is typically much thicker and some have lysine instead. An unusual feature of peptidoglycan is consists primarily of a single type of molecule. the presence of two amino acids of the D stereoisomer, D-alanine The focus of this section is on the polysaccharide component and D-glutamic acid. Proteins, by contrast, are always constructed of the cell walls of Bacteria, both gram-positive and gram-negative. of L-amino acids. In the next section we describe the special wall components present More than 100 different peptidoglycans are known, with diver- in gram-negative Bacteria. And finally, in Section 3.8 we briefly sity typically governed by the peptide cross-links and interbridge. describe the cell walls of Archaea. In every form of peptidoglycan the glycan portion is constant; only the sugars N-acetylglucosamine and N-acetylmuramic acid Peptidoglycan are present and are connected in β-1,4 linkage (Figure 3.16). The walls of Bacteria have a rigid layer that is primarily responsi- Moreover, the tetrapeptide shows major variation in only one ble for the strength of the wall. In gram-negative bacteria, addi- amino acid, the lysine–DAP alternation. Thus, although the tional layers are present outside this rigid layer. The rigid layer,  CHAPTER 3 Cell Structure and Function in Bacteria and Archaea 59 UNIT 1 Gram-positive Gram-negative Outer membrane Peptidoglycan Cytoplasmic membrane Protein Protein Leon J. Lebeau (a) (b) Peptidoglycan Cytoplasmic Cytoplasmic Peptidoglycan Outer membrane membrane membrane (c) (d) A.Umeda and K. Amako A.Umeda and K. Amako (e) (f) Figure 3.15 Cell walls of Bacteria. (a, b) Schematic diagrams of gram-positive and gram-negative cell walls. The Gram stain photo in the center shows cells of Staphylococcus aureus (purple, gram-positive) and Escherichia coli (pink, gram-negative). (c, d) Transmission electron micrographs (TEMs) showing the cell wall of a gram-positive bacterium and a gram-negative bacterium. (e, f) Scanning electron micrographs of gram-positive and gram-negative bacteria, respectively. Note differences in surface texture. Each cell in the TEMs is about 1 ␮m wide. peptide composition of peptidoglycan can vary, the peptidogly- peptidoglycan surrounding the cell, many gram-positive bacteria can backbone—alternating repeats of N-acetylglucosamine and have several sheets of peptidoglycan stacked one upon another N-acetylmuramic acid—is invariant. (Figure 3.15a). It is thought that the peptidoglycan is laid down by the cell in “cables” about 50 nm wide, with each cable consist- The Gram-Positive Cell Wall ing of several cross-linked glycan strands (Figure 3.18a). As the In gram-positive bacteria, as much as 90% of the wall is peptido- peptidoglycan “matures,” the cables themselves become cross- glycan. And, although some bacteria have only a single layer of linked to form an even stronger cell wall structure. 60 UNIT 1 Basic Principles of Microbiology N-Acetylglucosamine G N-Acetylmuramic acid M Polysaccharide backbone Interbridge CH2OH CH2OH G M G G M G O O H H L-Ala Peptides L-Ala Gly H H O O O D-Glu D-Glu-NH2 Gly ␤(1,4 OH H ␤(1,4 H ␤(1,4 ) H ) H ) DAP D-Ala L-Lys Gly H NH H NH D-Ala DAP D-Ala Gly Glycan tetrapeptide N-Acetyl C O O C O D-Glu Gly group HC CH3 CH3 CH3 L-Ala D-Ala C O Lysozyme- G M G L-Lys NH sensitive O bond Peptide (a) Escherichia coli D-Glu-NH2 H3C CH C cross-links (gram-negative) L-Ala NH L-Alanine O C CH2 CH2 CH COOH G M G Leon J. Lebeau NH2 NH D-Glutamic acid (b) Staphylococcus aureus O (gram-positive) HOOC C CH2 CH2 CH2 CH C Diaminopimelic H NH acid Y G M G M G M G M M G H3C CH COOH M G M G M G M G M M D-Alanine G G M M Peptide bonds M G M G M G M G M G G G M G M G M G M M G G M M Figure 3.16 Structure of the repeating unit in peptidoglycan, the G M G M G M M G G M M G G M G M G M G M glycan tetrapeptide. The structure given is that found in Escherichia coli M G G M G and most other gram-negative Bacteria. In some Bacteria, other amino acids are present as discussed in the text. X Glycosidic bonds (c) Many gram-positive bacteria have acidic components called Figure 3.17 Peptidoglycan in Escherichia coli and Staphylococcus teichoic acids embedded in their cell wall. The term “teichoic aureus. (a) No interbridge is present in E. coli peptidoglycan nor that of acids” includes all cell wall, cytoplasmic membrane, and capsular other gram-negative Bacteria. (b) The glycine interbridge in S. aureus polymers composed of glycerol phosphate or ribitol phosphate. (gram-positive). (c) Overall structure of peptidoglycan. G, N-acetylglu- These polyalcohols are connected by phosphate esters and typi- cosamine; M, N-acetylmuramic acid. Note how glycosidic bonds confer cally contain sugars or D-alanine (Figure 3.18b). Teichoic acids strength on peptidoglycan in the X direction whereas peptide bonds are covalently bonded to muramic acid in the wall peptidoglycan. confer strength in the Y direction. Because the phosphates are negatively charged, teichoic acids are at least in part responsible for the overall negative electrical charge of the cell surface. Teichoic acids also function to bind cytoplasmic membranes, and these probably function to add Ca21 and Mg21 for eventual transport into the cell. Certain tei- strength and rigidity to the membrane as they do in the cytoplas- choic acids are covalently bound to membrane lipids, and these mic membranes of eukaryotic cells. are called lipoteichoic acids (Figure 3.18c). Figure 3.18 summarizes the structure of the cell wall of gram- MiniQuiz positive Bacteria and shows how teichoic acids and lipoteichoic Why do bacterial cells need cell walls? Do all bacteria have cell acids are arranged in the overall wall structure. It also shows how walls? the peptidoglycan cables run perpendicular to the long axis of a Why is peptidoglycan such a strong molecule? rod-shaped bacterium. What does the enzyme lysozyme do? Cells That Lack Cell Walls Although most prokaryotes cannot survive in nature without 3.7 The Outer Membrane their cell walls, some do so naturally. These include the mycoplasmas, a group of pathogenic bacteria that causes several In gram-negative bacteria only about 10% of the total cell wall infectious diseases of humans and other animals, and the consists of peptidoglycan (Figure 3.15b). Instead, most of the wall Thermoplasma group, species of Archaea that naturally lack cell is composed of the outer membrane. This layer is effectively a walls. These bacteria are able to survive without cell walls second lipid bilayer, but it is not constructed solely of phospho- because they either contain unusually tough cytoplasmic mem- lipid and protein, as is the cytoplasmic membrane (Figure 3.5). branes or because they live in osmotically protected habitats The gram-negative cell outer membrane also contains polysac- such as the animal body. Most mycoplasmas have sterols in their charide. The lipid and polysaccharide are linked in the outer  CHAPTER 3 Cell Structure and Function in Bacteria and Archaea 61 Figure 3.18 Structure of the gram-positive bacterial cell wall. UNIT 1 (a) Schematic of a gram-positive rod showing the internal architecture of the peptidoglycan “cables.” (b) Structure of a ribitol teichoic acid. The teichoic acid is a polymer of the repeating ribitol unit shown here. (c) Summary diagram of the gram-positive bacterial cell wall. membrane to form a complex. Because of this, the outer mem- brane is also called the lipopolysaccharide layer, or simply LPS. Peptidoglycan cable Chemistry and Activity of LPS (a) The chemistry of LPS from several bacteria is known. As seen in D-Alanine D-Alanine D-Glucose O– O Figure 3.19, the polysaccharide portion of LPS consists of two P components, the core polysaccharide and the O-polysaccharide. O O O O O In Salmonella species, where LPS has been best studied, the Ribitol C C C C C core polysaccharide consists of ketodeoxyoctonate (KDO), vari- ous seven-carbon sugars (heptoses), glucose, galactose, and O N-acetylglucosamine. Connected to the core is the O-polysaccha- O P O– ride, which typically contains galactose, glucose, rhamnose, and O mannose, as well as one or more dideoxyhexoses, such as abequ- (b) ose, colitose, paratose, or tyvelose. These sugars are connected in four- or five-membered sequences, which often are branched. Wall-associated Teichoic acid Peptidoglycan Lipoteichoic protein acid When the sequences repeat, the long O-polysaccharide is formed. The relationship of the LPS layer to the overall gram-negative cell wall is shown in Figure 3.20. The lipid portion of the LPS, called lipid A, is not a typical glycerol lipid (see Figure 3.4a), but instead the fatty acids are connected through the amine groups from a disaccharide composed of glucosamine phosphate (Figure 3.19). The disaccharide is attached to the core polysaccharide through KDO (Figure 3.19). Fatty acids commonly found in lipid A include caproic (C6), lauric (C12), myristic (C14), palmitic (C16), and stearic (C18) acids. LPS replaces much of the phospholipid in the outer half of the outer membrane bilayer. By contrast, lipoprotein is present on the inner half of the outer membrane, along with the usual phos- pholipids (Figure 3.20a). Lipoprotein functions as an anchor tying the outer membrane to peptidoglycan. Thus, although the