Introduction

In this chapter we discuss the basic structure and function of the components that make up eukaryotic and prokaryotic cells. The chapter begins with a discussion of the microscope. Historically, it was the microscope that first revealed the presence of bacteria and later, the secrets of cell structure. Today, it remains a powerful tool in cell biology.

Optical Methods

The Light Microscope

The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used. (Resolving power is the distance that must separate two point sources of light if they are to be seen as two distinct images.) With yellow light of a wavelength of 0.4 m, the smallest separable diameters are thus about 0.2 m, ie, one-third the width of a typical prokaryotic cell. The useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles. Several types of light microscopes are commonly used in microbiology:

Bright-Field Microscope

The bright-field microscope is most commonly used in microbiology courses and consists of two series of lenses (objective and ocular lens), which function together to resolve the image. These microscopes generally employ a 100-power objective lens with a 10-power ocular lens, thus magnifying the specimen 1000 times. Particles 0.2 m in diameter are therefore magnified to about 0.2 mm and so become clearly visible. Further magnification would give no greater resolution of detail and would reduce the visible area (field).

With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium. Many bacteria are difficult to see well because of their lack of contrast with the surrounding medium. Dyes (stains) can be used to stain cells or their organelles and increase their contrast so that they can be more easily seen in the bright-field microscope.

Phase Contrast Microscope

The phase contrast microscope was developed to improve contrast differences between cells and the surrounding medium, making it possible to see living cells without staining them; with bright-field microscopes, killed and stained preparations must be used. The phase contrast microscope takes advantage of the fact that light waves passing through transparent objects, such as cells, emerge in different phases depending on the properties of the materials through which they pass. This effect is amplified by a special ring in the objective lens of a phase contrast microscope, leading to the formation of a dark image on a light background.

Dark-Field Microscope

The dark-field microscope is a light microscope in which the lighting system has been modified to reach the specimen from the sides only. This is accomplished through the use of a special condenser that both blocks direct light rays and deflects light off a mirror on the side of the condenser at an oblique angle. This creates a "dark field" that contrasts against the highlighted edge of the specimens and results when the oblique rays are reflected from the edge of the specimen upward into the objective of the microscope. Resolution by dark-field microscopy is quite high. Thus, this technique has been particularly useful for observing organisms such as Treponema pallidum, a spirochete which is less than 0.2 m in diameter and therefore cannot be observed with a bright-field or phase contrast microscope (Figure 2–1).

Figure 2–1.




Positive darkfield examination. Treponemes are recognized by their characteristic corkscrew shape and deliberate forward and backward movement with rotation about the longitudinal axis.

(Reproduced, with permission, from Morse SA, Moreland AA, Thompson SE [editors]: Atlas of Sexually Transmitted Disease. Gower, 1990.)



Fluorescence Microscope

The fluorescence microscope is used to visualize specimens that fluoresce, which is the ability to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). Some organisms fluoresce naturally because of the presence within the cells of naturally fluorescent substances such as chlorophyll. Those that do not naturally fluoresce may be stained with a group of fluorescent dyes called fluorochromes. Fluorescense microscopy is widely used in clinical diagnostic microbiology. For example, the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis, the bacterium that causes tuberculosis. When the dye is applied to a specimen suspected of containing M tuberculosis and exposed to ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms against a dark background.

The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent-antibody (FA) technique or immunofluorescence. In this technique, specific antibodies (eg, antibodies to Legionella pneumophila) are chemically labeled with a fluorochrome such as fluorescein isothiocyanate (FITC). These fluorescent antibodies are then added to a microscope slide containing a clinical specimen. If the specimen contains L pneumophila, the fluorescent antibodies will bind to antigens on the surface of the bacterium, causing it to fluoresce when exposed to ultraviolet light.

Differential Interference Contrast (DIC) Microscope

Differential interference contrast microscopes employ a polarizer to produce polarized light. The polarized light beam passes through a prism that generates two distinct beams; these beams pass through the specimen and enter the objective lens where they are recombined into a single beam. Because of slight differences in refractive index of the substances each beam passed through, the combined beams are not totally in phase but instead create an interference effect, which intensifies subtle differences in cell structure. Structures such as spores, vacuoles, and granules appear three dimensional. DIC microscopy is particularly useful for observing unstained cells because of its ability to generate images that reveal internal cell structures that are less apparent by bright-field techniques.

The Electron Microscope

The high resolving power of the electron microscope has enabled scientists to observe the detailed structures of prokaryotic and eukaryotic cells. The superior resolution of the electron microscope is due to the fact that electrons have a much shorter wavelength than the photons of white light.

There are two types of electron microscopes in general use: the transmission electron microscope (TEM), which has many features in common with the light microscope, and the scanning electron microscope (SEM). The TEM was the first to be developed and employs a beam of electrons projected from an electron gun and directed or focused by an electromagnetic condenser lens onto a thin specimen. As the electrons strike the specimen, they are differentially scattered by the number and mass of atoms in the specimen; some electrons pass through the specimen and are gathered and focused by an electromagnetic objective lens, which presents an image of the specimen to the projector lens system for further enlargement. The image is visualized by allowing it to impinge on a screen that fluoresces when struck with the electrons. The image can be recorded on photographic film. TEM can resolve particles 0.001 m apart. Viruses, with diameters of 0.01–0.2 m, can be easily resolved.

The SEM generally has a lower resolving power than the TEM; however, it is particularly useful for providing three-dimensional images of the surface of microscopic objects. Electrons are focused by means of lenses into a very fine point. The interaction of electrons with the specimen results in the release of different forms of radiation (eg, secondary electrons) from the surface of the material, which can be captured by an appropriate detector, amplified, and then imaged on a television screen.

An important technique in electron microscopy is the use of "shadowing." This involves depositing a thin layer of heavy metal (such as platinum) on the specimen by placing it in the path of a beam of metal ions in a vacuum. The beam is directed at a low angle to the specimen, so that it acquires a "shadow" in the form of an uncoated area on the other side. When an electron beam is then passed through the coated preparation in the electron microscope and a positive print is made from the "negative" image, a three-dimensional effect is achieved (eg, see Figure 2–22).

Figure 2–22.








Bacterial flagellation. A: Vibrio metchnikovii, a monotrichous bacterium (7500 x).

(Courtesy of van Iterson W: Biochim Biophys Acta 1947;1:527.)

B: Electron micrograph of Spirillum serpens, showing lophotrichous flagellation (9000 x).

(Courtesy of van Iterson W: Biochim Biophys Acta 1947;1:527.)

C: Electron micrograph of Proteus vulgaris, showing peritrichous flagellation (9000 x). Note basal granules.

(Courtesy of Houwink A, van Iterson W: Biochim Biophys Acta 1950;5:10.)



Other important techniques in electron microscopy include the use of ultrathin sections of embedded material, a method of freeze-drying specimens that prevents the distortion caused by conventional drying procedures, and the use of negative staining with an electron-dense material such as phosphotungstic acid or uranyl salts (eg, see Figure 42–1). Without these heavy metal salts, there would not be enough contrast to detect the details of the specimen.

Confocal Scanning Laser Microscope

The confocal scanning laser microscope (CSLM) couples a laser light source to a light microscope. In confocal scanning laser microscopy, a laser beam is bounced off a mirror that directs the beam through a scanning device. Then the laser beam is directed through a pinhole that precisely adjusts the plane of focus of the beam to a given vertical layer within the specimen. By precisely illuminating only a single plane of the specimen, illumination intensity drops off rapidly above and below the plane of focus, and stray light from other planes of focus are minimized. Thus, in a relatively thick specimen, various layers can be observed by adjusting the plane of focus of the laser beam.

Cells are often stained with fluorescent dyes to make them more visible. Alternatively, false color images can be generated by adjusting the microscope in such a way as to make different layers take on different colors. The CSLM is equipped with computer software to assemble digital images for subsequent image processing. Thus, images obtained from different layers can be stored and then digitally overlaid to reconstruct a three-dimensional image of the entire specimen.

Eukaryotic Cell Structure

The Nucleus

The nucleus contains the cell's genome. It is bounded by a membrane that consists of a pair of unit membranes separated by a space of variable thickness. The inner membrane is usually a simple sac, but the outermost membrane is, in many places, continuous with the endoplasmic reticulum. The nuclear membrane exhibits selective permeability due to pores, which consist of a complex of several proteins whose function is to import substances into and export substances out of the nucleus. The chromosomes of eukaryotic cells contain linear DNA macromolecules arranged as a double helix. They are only visible with a light microscope when the cell is undergoing division and the DNA is in a highly condensed form; at other times, the chromosomes are not condensed and appear as in Figure 2–2. Eukaryotic DNA macromolecules are associated with basic proteins called histones that bind to the DNA by ionic interactions.

Figure 2–2.




Electron micrograph of a thin section of a typical eukaryotic nucleus showing a prominent nucleolus and large aggregations of heterochromatin against the nuclear membrane, which is traversed by pores (at arrows). Inset upper left: Two nuclear pores and their pore diaphragms. Inset lower right: The fibrous lamina present in the inner aspect of the nuclear envelope. Several mitochondria are visible in the cytoplasm.

(Reproduced, with permission, from Fawcett DW: Bloom and Fawcett, A Textbook of Histology, 12th ed. Copyright © 1994. By permission of Chapman & Hall, New York, NY.)



A structure often visible within the nucleus is the nucleolus, an area rich in RNA that is the site of ribosomal RNA synthesis (Figure 2–2). Ribosomal proteins synthesized in the cytoplasm are transported into the nucleolus and combine with ribosomal RNA to form the small and large subunits of the eukaryotic ribosome. These are then exported to the cytoplasm where they associate to form an intact ribosome that can function in protein synthesis.

Cytoplasmic Structures

The cytoplasm of eukaryotic cells is characterized by the presence of an endoplasmic reticulum, vacuoles, self-reproducing plastids, and an elaborate cytoskeleton composed of microtubules, microfilaments, and intermediate filaments.

The endoplasmic reticulum (ER) is a network of membrane-bound channels continuous with the nuclear membrane. Two types of endoplasmic reticulum are recognized: rough, which contains attached 80S ribosomes, and smooth, which does not (Figure 2–2). Rough ER is a major producer of glycoproteins and also produces new membrane material that is transported throughout the cell; smooth ER participates in the synthesis of lipids and in some aspects of carbohydrate metabolism. The Golgi apparatus consists of a stack of membranes that function in concert with the ER to chemically modify and sort products of the ER into those destined to be secreted and those that function in other membranous structures of the cell.

The plastids include mitochondria and chloroplasts. Several lines of evidence suggest that mitochondria and chloroplasts were descendents of ancient prokaryotic organisms and arose from the engulfment of a prokaryotic cell by a larger cell (endosymbiosis). Mitochondria are of prokaryotic size, and its membrane, which lacks sterols, is much less rigid than the eukaryotic cell's cytoplasmic membrane, which does contain sterols. Mitochondria contain two sets of membranes. The outermost membrane is rather permeable having numerous minute channels that allow passage of ions and small molecules (eg, ATP). Invagination of the outer membrane forms a system of inner folded membranes called cristae. The cristae are the sites of enzymes involved in respiration and ATP production. Cristae also contain specific transport proteins that regulate passage of metabolites into and out of the mitochondrial matrix. The matrix contains a number of enzymes, in particular those of the citric acid cycle. Chloroplasts are photosynthetic cell organelles that are capable of converting the energy of sunlight into chemical energy through photosynthesis. Chlorophyll and all other components needed for photosynthesis are located in a series of flattened membrane discs called thylakoids. The size, shape, and number of chloroplasts per cell vary markedly; in contrast to mitochondria, chloroplasts are generally much larger than prokaryotes. Mitochondria and chloroplasts contain their own DNA, which exists in a covalently closed circular form and codes for some (not all) of their constituent proteins and transfer RNAs. Mitochondria and chloroplasts also contain 70S ribosomes, the same as those of prokaryotes.

Some eukaryotic microorganisms (eg, Trichomonas vaginalis) lack mitochondria and contain instead a membrane-enclosed respiratory organelle called the hydrogenosome. Hydrogenosomes may have arisen by endosymbiosis and some have been identified that contain DNA and ribosomes. The hydrogenosome, while similar in size to mitochondria, lacks cristae and the enzymes of the tricarboxylic acid cycle. Pyruvate is taken up by the hydrogenosome and H2, CO2, acetate, and ATP are produced.

Lysosomes are membrane-enclosed sacs that contain various digestive enzymes that the cell uses to digest macromolecules such as proteins, fats, and polysaccharides. The lysosome allows these enzymes to be partitioned away from the cytoplasm proper where they could destroy key cellular macromolecules if not contained. Following the hydrolysis of macromolecules in the lysosome, the resulting monomers pass from the lysosome into the cytoplasm where they serve as nutrients.

The peroxisome is a membrane-enclosed structure whose function is to produce H2O2 from the reduction of O2 by various hydrogen donors. The H2O2 produced in the peroxisome is subsequently degraded to H2O and O2 by the enzyme catalase.

The cytoskeleton is a three-dimensional structure that fills the cytoplasm. The primary types of fibers comprising the cytoskeleton are microfilaments, intermediate filaments, and microtubules. Microfilaments are about 3–6 nm in diameter and are polymers composed of subunits of the protein actin. These fibers form scaffolds throughout the cell defining and maintaining the shape of the cell. Microfilaments can also carry out cellular movements including gliding, contraction, and cytokinesis.

Microtubules are cylindrical tubes, 20–25 nm in diameter and are composed of subunits of the protein tubulin. Microtubules assist microfilaments in maintaining cell structure, form the spindle fibers for separating chromosomes during mitosis, and also play an important role in cell motility. Intermediate filaments are about 10 nm in diameter and provide tensile strength for the cell.

Surface Layers

The cytoplasm is enclosed within a plasma membrane composed of protein and phospholipid, similar to the prokaryotic cell membrane illustrated later (see Figure 2–10). Most animal cells have no other surface layers; however, plant cells have an outer cell wall composed of cellulose. Many eukaryotic microorganisms also have an outer cell wall, which may be composed of a polysaccharide such as cellulose or chitin or may be inorganic, eg, the silica wall of diatoms.

Figure 2–10.




A model of membrane structure. Folded polypeptide molecules are visualized as embedded in a phospholipid bilayer, with their hydrophilic regions protruding into the intracellular space, extracellular space, or both.

(Reproduced, with permission, from Singer SJ, Nicolson AL: The fluid mosaic model of the structure of cell membranes. Science 1972;175:720. Copyright © 1972 by the American Association for the Advancement of Science.)



Motility Organelles

Many eukaryotic microorganisms have organelles called flagella (eg, Trichomonas vaginalis) or cilia (eg, Balantidium coli) that move with a wave-like motion to propel the cell through water. Eukaryotic flagella emanate from the polar region of the cell, whereas cilia, which are shorter than flagella, surround the cell. Both the flagella and the cilia of eukaryotic cells have the same basic structure and biochemical composition. Both consist of a series of microtubules, hollow protein cylinders composed of a protein called tubulin, surrounded by a membrane. The arrangement of the microtubules is called the "9 + 2 system" because it consists of nine peripheral pairs of microtubules surrounding two single central microtubules (Figure 2–3).

Figure 2–3.





Fine structure of eukaryotic flagella and cilia (31,500 x). A: Cross section through surface layer of the ciliate protozoan glaucoma, which cuts across a field of cilia just within the cell membrane (lower half) as well as outside the cell membrane (upper half). (cm, cell membrane; cs, cell surface; if, inner fibrils; of, outer fibrils) B: Diagram of the parts of a flagellum or cilium. The various projections from the microtubules link the microtubules together and occur at regular intervals along the length of the axoneme.

(A, Electron micrograph taken by D. Pitelka. Reproduced, with permission, from Stanier RY, Doudoroff M, Adelberg EA: The Microbial World, 2nd ed. Copyright ©1963. By permission of Prentice-Hall, Inc., Englewood Cliffs, NJ. B, Reproduced, with permission, from Roberts A et al: Molecular Biology of the Cell, 4th ed. Garland Science, 2002.)



Prokaryotic Cell Structure

The prokaryotic cell is simpler than the eukaryotic cell at every level, with one exception: The cell envelope is more complex.

The Nucleoid

Prokaryotes have no true nuclei; instead they package their DNA in a structure known as the nucleoid. The nucleoid can be seen with the light microscope in stained material (Figure 2–4). It is Feulgen-positive, indicating the presence of DNA. The negatively charged DNA is at least partially neutralized by small polyamines and magnesium ions, but histone-like proteins exist in bacteria and presumably play a role similar to that of histones in eukaryotic chromatin.

Figure 2–4.




Nucloids of Bacillus cereus (2500 x).

(Courtesy of Robinow C: Bacteriol Rev 1956;20:207.)




Electron micrographs of a typical prokaryotic cell such as Figure 2–5 reveal the absence of a nuclear membrane and a mitotic apparatus. The exception to this rule is the planctomycetes, a divergent group of aquatic bacteria, which have a nucleoid surrounded by a nuclear envelope consisting of two membranes. The distinction between prokaryotes and eukaryotes that still holds is that prokaryotes have no eukaryotic-type mitotic apparatus. The nuclear region (Figure 2–5) is filled with DNA fibrils. The nucleoid of most bacterial cells consists of a single continuous circular molecule ranging in size from 0.58 to almost 10 million base pairs. However, a few bacteria have been shown to have two, three, or even four dissimilar chromosomes. For example, Vibrio cholerae and Brucella melitensis have two dissimilar chromosomes. There are exceptions to this rule of circularity because some prokaryotes (eg, Borrelia burgdorferi and Streptomyces coelicolor) have been shown to have a linear chromosome.

Figure 2–5.




Thin section of E coli cell fixed with osmium tetroxide and postfixed with aqueous uranyl acetate showing two nuclear regions filled with DNA fibrils.

(Courtesy of Robinow C, Kellenberger E: Microbiol Rev 1994;58:211.)



In bacteria, the number of nucleoids, and therefore the number of chromosomes, depends on the growth conditions (Figure 2–4). Rapidly growing bacteria have more nucleoids per cell than slowly growing ones; however, when multiple copies are present they are all the same (ie, prokaryotic cells are haploid).

Cytoplasmic Structures

Prokaryotic cells lack autonomous plastids, such as mitochondria and chloroplasts; the electron transport enzymes are localized instead in the cytoplasmic membrane. The photosynthetic pigments (carotenoids, bacteriochlorophyll) of photosynthetic bacteria are contained in intracytoplasmic membrane systems of various morphologies. Membrane vesicles (chromatophores) or lamellae are commonly observed membrane types. Some photosynthetic bacteria have specialized non-unit membrane-enclosed structures called chlorosomes. In some cyanobacteria (formerly known as blue-green algae), the photosynthetic membranes often form multilayered structures known as thylakoids (Figure 2–6). The major accessory pigments used for light harvesting are the phycobilins found on the outer surface of the thylakoid membranes.

Figure 2–6.




Thin section of a cyanobacterium, anacystis (80,500 x). (l, lamellae bearing photosynthetic pigments; cw, cell wall; n, nuclear region.)

(Reprinted by permission of the Rockefeller Institute Press, from Ris H, Singh RN: J Biophys Biochem Cytol 1961;9:63.)




Bacteria often store reserve materials in the form of insoluble granules, which appear as refractile bodies in the cytoplasm when viewed in a phase contrast microscope. These so-called inclusion bodies almost always function in the storage of energy or as a reservoir of structural building blocks. Most cellular inclusions are bounded by a thin nonunit membrane consisting of lipid, which serves to separate the inclusion from the cytoplasm proper. One of the most common inclusion bodies consists of poly--hydroxybutyric acid (PHB), a lipid-like compound consisting of chains of -hydroxybutyric acid units connected through ester linkages. PHB is produced when the source of nitrogen, sulfur, or phosphorous is limited and there is excess carbon in the medium (Figure 2–7). Another storage product formed by prokaryotes when carbon is in excess is glycogen, which is a polymer of glucose. PHB and glycogen are used as carbon sources when protein and nucleic acid synthesis are resumed. A variety of prokaryotes are capable of oxidizing reduced sulfur compounds such as hydrogen sulfide and thiosulfate, producing intracellular granules of elemental sulfur. As the reduced sulfur source becomes limiting, the sulfur in the granules is oxidized, usually to sulfate, and the granules slowly disappear. Many bacteria accumulate large reserves of inorganic phosphate in the form of granules of polyphosphate. These granules can be degraded and used as sources of phosphate for nucleic acid and phospholipid synthesis to support growth. These granules are sometimes termed volutin granules or metachromatic granules because they stain red with a blue dye. They are characteristic features of the corynebacteria (Chapter 13).

Figure 2-7.




Formation and utilization of poly--hydroxybutyric acid in Bacillus megaterium (1900x). Left: Cells grown on glucose plus acetate, showing granules (light areas). Right: Cells from the same culture after 24 hours' further incubation in the presence of a nitrogen source but without an exogenous carbon source. The polymer has been completely metabolized. Phase contrast photomicrograph taken by Dr JF Wilkinson.




Certain groups of autotrophic bacteria that fix carbon dioxide to make their biochemical building blocks contain polyhedral bodies surrounded by a protein shell (carboxysomes) containing the key enzyme of CO2 fixation, ribulosebisphosphate carboxylase. Magnetosomes are intracellular crystal particles of the iron mineral magnetite (Fe3O4) that allow certain aquatic bacteria to exhibit magnetotaxis (ie, migration or orientation of the cell with respect to the earth's magnetic field). Magnetosomes are surrounded by a nonunit membrane containing phospholipids, proteins, and glycoproteins. Gas vesicles are found almost exclusively in microorganisms from aquatic habitats, where they provide buoyancy. The gas vesicle membrane is a 2 nm thick layer of protein, impermeable to water and solutes but permeable to gases; thus, gas vesicles exist as gas-filled structures surrounded by the constituents of the cytoplasm (Figure 2–8).

Figure 2–8.




Transverse section of a dividing cell of the cyanobacterium microcystis species showing hexagonal stacking of the cylindric gas vesicles (31,500x).

(Micrograph by HS Pankratz. Reproduced, with permission, from Walsby AE: Gas vesicles. Microbiol Rev 1994;58:94.)



Bacteria contain proteins resembling both the actin and nonactin cytoskeletal proteins of eukaryotic cells as additional proteins that play cytoskeletal roles. Actin homologs (eg, MreB, Mbl) perform a variety of functions, helping to determine cell shape, segregate chromosomes, and localize proteins with the cell. Nonactin homologs (eg, FtsZ) and unique bacterial cytoskeletal proteins (eg, SecY, MinD) are involved in determining cell shape and in regulation of cell division and chromosome segregation.

The Cell Envelope

Prokaryotic cells are surrounded by complex envelope layers that differ in composition among the major groups. These structures protect the organisms from hostile environments, such as extreme osmolarity, harsh chemicals, and even antibiotics.

The Cell Membrane

Structure

The bacterial cell membrane, also called the cytoplasmic membrane, is visible in electron micrographs of thin sections (Figure 2–9). It is a typical "unit membrane" composed of phospholipids and upward of 200 different kinds of proteins. Proteins account for approximately 70% of the mass of the membrane, which is a considerably higher proportion than that of mammalian cell membranes. Figure 2–10 illustrates a model of membrane organization. The membranes of prokaryotes are distinguished from those of eukaryotic cells by the absence of sterols, the only exception being mycoplasmas that incorporate sterols, such as cholesterol, into their membranes when growing in sterol-containing media.

Figure 2–9.




The cell membrane. Fragments of the cell membrane (CM) are seen attached to the cell wall (CW) in preparations made from Escherichia coli.

(Reproduced, with permission, from Schnaitman CA: Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol 1971;108:545.)



The cell membranes of the Archaea (see Chapter 1) differ from those of the Bacteria. Some Archaeal cell membranes contain unique lipids, isoprenoids, rather than fatty acids, linked to glycerol by an ether rather than an ester linkage. Some of these lipids have no phosphate groups, and therefore, they are not phospholipids. In other species the cell membrane is made up of a lipid monolayer consisting of long lipids (about twice as long as a phospholipid) with glycerol ethers at both ends (diglycerol tetraethers). The molecules orient themselves with the polar glycerol groups on the surfaces and the nonpolar hydrocarbon chain in the interior. These unusual lipids contribute to the ability of many Archaea to grow under environmental conditions such as high salt, low pH, or very high temperature.

Function

The major functions of the cytoplasmic membrane are (1) selective permeability and transport of solutes; (2) electron transport and oxidative phosphorylation, in aerobic species; (3) excretion of hydrolytic exoenzymes; (4) bearing the enzymes and carrier molecules that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids; and (5) bearing the receptors and other proteins of the chemotactic and other sensory transduction systems.

At least 50% of the cytoplasmic membrane must be in the semifluid state in order for cell growth to occur. At low temperatures, this is achieved by greatly increased synthesis and incorporation of unsaturated fatty acids into the phospholipids of the cell membrane.

Permeability and Transport

The cytoplasmic membrane forms a hydrophobic barrier impermeable to most hydrophilic molecules. However, several mechanisms (transport systems) exist that enable the cell to transport nutrients into and waste products out of the cell. These transport systems work against a concentration gradient to increase the concentration of nutrients inside the cell, a function that requires energy in some form. There are three general transport mechanisms involved in membrane transport: passive transport, active transport, and group translocation.

Passive Transport

This mechanism relies on diffusion, uses no energy, and operates only when the solute is at higher concentration outside than inside the cell. Simple diffusion accounts for the entry of very few nutrients including dissolved oxygen, carbon dioxide, and water itself. Simple diffusion provides neither speed nor selectivity. Facilitated diffusion also uses no energy so the solute never achieves an internal concentration greater than what exists outside the cell. However, facilitated diffusion is selective. Channel proteins form selective channels that facilitate the passage of specific molecules. Facilitated diffusion is common in eukaryotic microorganisms (eg, yeast), but is rare in prokaryotes. Glycerol is one of the few compounds that enters prokaryotic cells by facilitated diffusion.

Active Transport

Many nutrients are concentrated more than a thousand fold as a result of active transport. There are two types of active transport mechanisms depending upon the source of energy employed: ion-coupled transport and ATP-binding cassette (ABC) transport.

Ion-Coupled Transport

These systems move a molecule across the cell membrane at the expense of a previously established ion gradient such as proton-motive or sodium-motive force. There are three basic types: uniport, symport, and antiport (Figure 2–11). Ion-coupled transport is particularly common in aerobic organisms, which have an easier time generating an ion-motive force than do anaerobes. Uniporters catalyze the transport of a substrate independent of any coupled ion. Symporters catalyze the simultaneous transport of two substrates in the same direction by a single carrier; for example, an H+ gradient can permit symport of an oppositely charged ion (eg, glycine) or a neutral molecule (eg, galactose). Antiporters catalyze the simultaneous transport of two like-charged compounds in opposite directions by a common carrier (eg, H+:Na+). Approximately 40% of the substrates transported by Escherichia coli utilize this mechanism.

Figure 2–11.




Three types of porters: uniporters (top), symporters (middle), and antiporters (bottom). Uniporters catalyze the transport of a single species independently of any other, symporters catalyze the cotransport of two dissimilar species (usually a solute and a positively charged ion, H+) in the same direction, and antiporters catalyze the exchange transport of two similar solutes in opposite directions. A single transport protein may catalyze just one of these processes, two of these processes, or even all three of these processes, depending on conditions. Uniporters, symporters, and antiporters have been found to be structurally similar and evolutionarily related, and they function by similar mechanisms.

(Reproduced, with permission, from Saier MH Jr: Peter Mitchell and his chemiosmotic theories. ASM News 1997;63:13.)



ABC Transport

This mechanism employs ATP directly to transport solutes into the cell. In gram-negative bacteria, the transport of many nutrients is facilitated by specific binding proteins located in the periplasmic space; in gram-positive cells the binding proteins are attached to the outer surface of the cell membrane. These proteins function by transferring the bound substrate to a membrane-bound protein complex. Hydrolysis of ATP is then triggered, and the energy is used to open the membrane pore and allow the unidirectional movement of the substrate into the cell. Approximately 40% of the substrates transported by E coli utilize this mechanism.

Group Translocation

In addition to true transport, in which a solute is moved across the membrane without change in structure, bacteria use a process called group translocation (vectorial metabolism) to effect the net uptake of certain sugars (eg, glucose and mannose), the substrate becoming phosphorylated during the transport process. In a strict sense, group translocation is not active transport because no concentration gradient is involved. This process allows bacteria to utilize their energy resources efficiently by coupling transport with metabolism. In this process, a membrane carrier protein is first phosphorylated in the cytoplasm at the expense of phosphoenolpyruvate; the phosphorylated carrier protein then binds the free sugar at the exterior membrane face and transports it into the cytoplasm, releasing it as sugar-phosphate. Such systems of sugar transport are called phosphotransferase systems. Phosphotransferase systems are also involved in movement towards these carbon sources (chemotaxis) and in the regulation of several other metabolic pathways (catabolite repression).

Special Transport Processes

Iron (Fe) is an essential nutrient for the growth of almost all bacteria. Under anaerobic conditions, Fe is generally in the +2 oxidation state and soluble. However, under aerobic conditions, Fe is generally in the +3 oxidation state and insoluble. The internal compartments of animals contain virtually no free Fe; it is sequestered in complexes with such proteins as transferrin and lactoferrin. Some bacteria solve this problem by secreting siderophores—compounds that chelate Fe and promote its transport as a soluble complex. One major group of siderophores consists of derivatives of hydroxamic acid (–CONH2OH), which chelate Fe3+ very strongly. The iron-hydroxamate complex is actively transported into the cell by the cooperative action of a group of proteins that span the outer membrane, periplasm, and inner membrane. The iron is released, and the hydroxamate can exit the cell and be used again for iron transport.

Some pathogenic bacteria use a fundamentally different mechanism involving specific receptors that bind host transferrin and lactoferrin (as well as other iron-containing host proteins). The Fe is removed and transported into the cell by an energy-dependent process.

Electron Transport and Oxidative Phosphorylation

The cytochromes and other enzymes and components of the respiratory chain, including certain dehydrogenases, are located in the cell membrane. The bacterial cell membrane is thus a functional analog of the mitochondrial membrane—a relationship which has been taken by many biologists to support the theory that mitochondria have evolved from symbiotic bacteria. The mechanism by which ATP generation is coupled to electron transport is discussed in Chapter 6.

Excretion of Hydrolytic Exoenzymes and Pathogenicity Proteins

All organisms that rely on macromolecular organic polymers as a source of nutrients (eg, proteins, polysaccharides, lipids) excrete hydrolytic enzymes that degrade the polymers to subunits small enough to penetrate the cell membrane. Higher animals secrete such enzymes into the lumen of the digestive tract; bacteria (both gram-positive and gram-negative) secrete them directly into the external medium or into the periplasmic space between the peptidoglycan layer and the outer membrane of the cell wall in the case of gram-negative bacteria (see The Cell Wall, below).

In gram-positive bacteria, proteins are secreted directly, but proteins secreted by gram-negative bacteria must traverse the outer membrane as well. Five pathways of protein secretion have been described in gram-negative bacteria: the type I, type II, type III, type IV, and type V secretion systems. A schematic overview of the type I, type II, and type III systems is presented in Figure 2–12. Proteins secreted by the type I and type III pathways traverse the inner membrane (IM) and outer membrane (OM) in one step, whereas proteins secreted by the type II and type V pathways cross the IM and OM in separate steps. Proteins secreted by the type II and type V pathways are synthesized on cytoplasmic ribosomes as preproteins containing an extra leader or signal sequence of 15 to 40 amino acids—most commonly about 30 amino acids—at the amino terminal and require the sec system for transport across the IM. In E coli, the sec pathway comprises a number of IM proteins (SecD to SecF, SecY), a cell membrane-associated ATPase (SecA) that provides energy for export, a chaperone (SecB) that binds to the preprotein, and the periplasmic signal peptidase. Following translocation, the leader sequence is cleaved off by the membrane-bound signal peptidase and the mature protein is released into the periplasmic space. In contrast, proteins secreted by the type I and type III systems do not have a leader sequence and are exported intact.

Figure 2–12.




Schematic overview of the type I, II, and III secretion systems as exemplified by alpha-hemolysin secretion by E coli (type I), pullulanase secretion by Klebsiella oxytoca (type II), and Yop secretion by yersinia (type III). OM, outer membrane; PP, periplasm; IM, inner membrane; CP, cytoplasm. ATP hydrolysis by HIyB, SecA, and YscN is indicated. The localization of the secretion signals is shown in the secreted proteins (dark lines). N, amino terminal; C, carboxyl terminal. For type III secretion, the secretion signal may reside in the 5' region of the mRNA encoding the secreted protein. Type II and type III secretions involve cytoplasmic chaperones (SecB and Syc, respectively), which bind to presecretory proteins. In type II secretion, the amino terminal signal sequence is cleaved off by a periplasmic peptidase (LspA) after export of the protein via the sec pathway. Type II and type III secretions share a homologous multimeric outer membrane component (PulD, YscC), while the accessory proteins S and VirG, which facilitate outer membrane insertion of PulD and YscC, respectively, differ in the two systems.

(Courtesy of Hueck CJ: Microbiol Mol Biol Rev 1998;62:379.)



Although proteins secreted by the type II and type V systems are similar in the mechanism by which they cross the IM, differences exist in how they traverse the OM. Proteins secreted by the type II system are transported across the OM by a multiprotein complex (Figure 2–12). This is the primary pathway for the secretion of extracellular degradative enzymes by gram-negative bacteria. Elastase, phospholipase C, and exotoxin A are secreted by this system in Pseudomonas aeruginosa. However, proteins secreted by the type V system autotransport across the outer membrane by virtue of a carboxyl terminal sequence which is enzymatically removed upon release of the protein from the OM. Some extracellular proteins—eg, the IgA protease of Neisseria gonorrhoeae and the vacuolating cytotoxin of Helicobacter pylori—are secreted by this system.

The type I and type III secretion pathways are sec-independent and thus do not involve amino terminal processing of the secreted proteins. Protein secretion by these pathways occurs in a continuous process without the presence of a cytoplasmic intermediate. Type I secretion is exemplified by the -hemolysin of E coli and the adenylyl cyclase of Bordetella pertussis. Type I secretion requires three secretory proteins: an IM ATP-binding cassette (ABC transporter), which provides energy for protein secretion; an OM protein; and a membrane fusion protein, which is anchored in the inner membrane and spans the periplasmic space (Figure 2–12). Instead of a signal peptide, the information is located within the carboxyl terminal 60 amino acids of the secreted protein.

The type III secretion pathway is a contact-dependent system. It is activated by contact with a host cell, and then injects a toxin protein into the host cell directly. The type III secretion apparatus is composed of approximately 20 proteins, most of which are located in the IM. Most of these IM components are homologous to the flagellar biosynthesis apparatus of both gram-negative and gram-positive bacteria. As in type I secretion, the proteins secreted via the type III pathway are not subject to amino terminal processing during secretion.

Type IV pathways secrete either polypeptide toxins (directed against eukaryotic cells) or protein-DNA complexes either between two bacterial cells or between a bacterial and a eukaryotic cell. Type IV secretion is exemplified by the protein-DNA complex delivered by Agrobacterium tumefaciens into a plant cell. Additionally, B pertussis and H pylori possess type IV secretion systems that mediate secretion of pertussis toxin and interleukin-8-inducing factor, respectively.

Biosynthetic Functions

The cell membrane is the site of the carrier lipids on which the subunits of the cell wall are assembled (see the discussion of synthesis of cell wall substances in Chapter 6) as well as of the enzymes of cell wall biosynthesis. The enzymes of phospholipid synthesis are also localized in the cell membrane.

Chemotactic Systems

Attractants and repellents bind to specific receptors in the bacterial membrane (see Flagella below). There are at least 20 different chemoreceptors in the membrane of E coli, some of which also function as a first step in the transport process.

The Cell Wall

The internal osmotic pressure of most bacteria ranges from 5 atm to 20 atm as a result of solute concentration via active transport. In most environments, this pressure would be sufficient to burst the cell were it not for the presence of a high-tensile-strength cell wall (Figure 2–13). The bacterial cell wall owes its strength to a layer composed of a substance variously referred to as murein, mucopeptide, or peptidoglycan (all are synonyms). The structure of peptidoglycan will be discussed below.

Figure 2–13.




Cell walls of Streptococcus faecalis, removed from protoplasts by mechanical disintegration and differential centrifugation (11,000 x).

(Courtesy of Salton M, Home R: Biochim Biophys Acta 1951;7:177.)




Most bacteria are classified as gram-positive or gram-negative according to their response to the Gram staining procedure. This procedure was named for the histologist Hans Christian Gram, who developed this differential staining procedure in an attempt to stain bacteria in infected tissues. The Gram stain depends on the ability of certain bacteria (the gram-positive bacteria) to retain a complex of crystal violet (a purple dye) and iodine after a brief wash with alcohol or acetone. Gram-negative bacteria do not retain the dye-iodine complex and become translucent, but they can then be counterstained with safranin (a red dye). Thus, gram-positive bacteria look purple under the microscope, and gram-negative bacteria look red. The distinction between these two groups turns out to reflect fundamental differences in their cell envelopes (Figure 2–14).

Figure 2–14.




Comparison of the structures of gram-positive and gram-negative cell envelopes. The region between the cytoplasmic membrane and the outer membrane of the gram-negative envelope is called the periplasmic space.

(Reproduced, with permission, from Ingraham JL, Maaløe O, Neidhardt FC: Growth of the Bacterial Cell. Sinauer Associates, 1983.)



In addition to giving osmotic protection, the cell wall plays an essential role in cell division as well as serving as a primer for its own biosynthesis. Various layers of the wall are the sites of major antigenic determinants of the cell surface, and one component—the lipopolysaccharide of gram-negative cell walls—is responsible for the nonspecific endotoxin activity of gram-negative bacteria. The cell wall is, in general, nonselectively permeable; one layer of the gram-negative wall, however—the outer membrane—hinders the passage of relatively large molecules (see below).

The biosynthesis of the cell wall and the antibiotics that interfere with this process are discussed in Chapter 6.

The Peptidoglycan Layer

Peptidoglycan is a complex polymer consisting, for the purposes of description, of three parts: a backbone, composed of alternating N-acetylglucosamine and N-acetylmuramic acid; a set of identical tetrapeptide side chains attached to N-acetylmuramic acid; and a set of identical peptide cross-bridges (Figure 2–15). The backbone is the same in all bacterial species; the tetrapeptide side chains and the peptide cross-bridges vary from species to species, those of Staphylococcus aureus being illustrated in Figure 2–15. In many gram-negative cell walls, the cross-bridge consists of a direct peptide linkage between the diaminopimelic acid (DAP) amino group of one side chain and the carboxyl group of the terminal D-alanine of a second side chain.

Figure 2–15.




A: A segment of the peptidoglycan of Staphylococcus aureus. The backbone of the polymer consists of alternating subunits of N-acetylglucosamine and N-acetylmuramic acid connected by 14 linkages. The muramic acid residues are linked to short peptides, the composition of which varies from one bacterial species to another. In some species, the L-lysine residues are replaced by diaminopimelic acid, an amino acid that is found in nature only in prokaryotic cell walls. Note the D-amino acids, which are also characteristic constituents of prokaryotic cell walls. The peptide chains of the peptidoglycan are cross-linked between parallel polysaccharide backbones, as shown in Figure 2–15B. B: Schematic representation of the peptidoglycan lattice that is formed by cross-linking. Bridges composed of pentaglycine peptide chains connect the -carboxyl of the terminal D-alanine residue of one chain with the -amino group of the L-lysine residue of the next chain. The nature of the cross-linking bridge varies among different species.



The tetrapeptide side chains of all species, however, have certain important features in common. Most have L-alanine at position 1 (attached to N-acetylmuramic acid), D-glutamate or substituted D-glutamate at position 2, and D-alanine at position 4. Position 3 is the most variable one: Most gram-negative bacteria have diaminopimelic acid at this position, to which is linked the lipoprotein cell wall component discussed below. Gram-positive bacteria usually have L-lysine at position 3; however, some may have diaminopimelic acid or another amino acid at this position.

Diaminopimelic acid is a unique element of bacterial cell walls. It is never found in the cell walls of Archaea or eukaryotes. Diaminopimelic acid is the immediate precursor of lysine in the bacterial biosynthesis of that amino acid (see Figure 6–18). Bacterial mutants that are blocked prior to diaminopimelic acid in the biosynthetic pathway grow normally when provided with diaminopimelic acid in the medium; when given L-lysine alone, however, they lyse, since they continue to grow but are specifically unable to make new cell wall peptidoglycan.

The fact that all peptidoglycan chains are cross-linked means that each peptidoglycan layer is a single giant molecule. In gram-positive bacteria, there are as many as 40 sheets of peptidoglycan, comprising up to 50% of the cell wall material; in gram-negative bacteria, there appears to be only one or two sheets, comprising 5–10% of the wall material. Bacteria owe their shapes, which are characteristic of particular species, to their cell wall structure.

Special Components of Gram-Positive Cell Walls

Most gram-positive cell walls contain considerable amounts of teichoic and teichuronic acids, which may account for up to 50% of the dry weight of the wall and 10% of the dry weight of the total cell. In addition, some gram-positive walls may contain polysaccharide molecules.

Teichoic and Teichuronic Acids

The term teichoic acids encompasses all wall, membrane, or capsular polymers containing glycerophosphate or ribitol phosphate residues. These polyalcohols are connected by phosphodiester linkages and usually have other sugars and D-alanine attached (Figure 2–16A). Because they are negatively charged, teichoic acids are partially responsible for the negative charge of the cell surface as a whole. There are two types of teichoic acids: wall teichoic acid (WTA), covalently linked to peptidoglycan, and membrane teichoic acid, covalently linked to membrane glycolipid. Because the latter are intimately associated with lipids, they have been called lipoteichoic acids (LTA). Together with peptidoglycan, WTA and LTA make up a polyanionic network or matrix that provides functions relating to the elasticity, porosity, tensile strength, and electrostatic properties of the envelope. Although not all gram-positive bacteria have conventional LTA and WTA, those that lack these polymers generally have functionally similar ones.

Figure 2–16A.




Repeat units of some teichoic acids. A: Glycerol teichoic acid of Lactobacillus casei 7469 (R, D-alanine). B: Glycerol teichoic acid of Actinomyces antibioticus (R, D-alanine). C: Glycerol teichoic acid of Staphylococcus lactis 13. D-Alanine occurs on the 6 position of N-acetylglucosamine. D: Ribitol teichoic acids of Bacillus subtilis (R, glucose) and Actinomyces streptomycini (R, succinate). (The D-alanine is attached to position 3 or 4 of ribitol.) E: Ribitol teichoic acid of the type 6 pneumococcal capsule. (Reproduced, with permission, from Stanier RY, Doudoroff M, Adelberg EA: The Microbial World, 2nd ed. Copyright © 1963. By permission of Prentice-Hall, Inc., Englewood Cliffs, NJ.)




The repeat units of some teichoic acids are shown in Figure 2–16A. The repeat units may be glycerol, joined by 1,3- or 1,2-linkages; ribitol, joined by 1,5-linkages, or more complex units in which glycerol or ribitol is joined to a sugar residue such as glucose, galactose, or N-acetylglucosamine. The chains may be 30 or more repeat units in length, though chain lengths of ten or less are common.

Most teichoic acids contain large amounts of D-alanine, usually attached to position 2 or 3 of glycerol or position 3 or 4 of ribitol. In some of the more complex teichoic acids, however, D-alanine is attached to one of the sugar residues. In addition to D-alanine, other substituents may be attached to the free hydroxyl groups of glycerol and ribitol, eg, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, or succinate. A given species may have more than one type of sugar substituent in addition to D-alanine; in such cases, it is not certain whether the different sugars occur on the same or on separate teichoic acid molecules. The composition of the teichoic acid formed by a given bacterial species can vary with the composition of the growth medium.

The teichoic acids constitute major surface antigens of those gram-positive species that possess them, and their accessibility to antibodies has been taken as evidence that they lie on the outside surface of the peptidoglycan. Their activity is often increased, however, by partial digestion of the peptidoglycan; thus, much of the teichoic acid may lie between the cytoplasmic membrane and the peptidoglycan layer, possibly extending upward through pores in the latter (Figure 2–16B). In the pneumococcus (Streptococcus pneumoniae), the teichoic acids bear the antigenic determinants called Forssman antigen. In Streptococcus pyogenes, LTA is associated with the M protein that protrudes from the cell membrane through the peptidoglycan layer. The long M protein molecules together with the LTA form microfibrils that facilitate the attachment of S pyogenes to animal cells.

Figure 2–16B.




Lipoteichoic acids. A model of the cell wall and membrane of a gram-positive bacterium, showing lipoteichoic acid molecules extending through the cell wall. The wall teichoic acids, covalently linked to muramic acid residues of the peptidoglycan layer, are not shown. (A, cell wall; B, protein; C, phospholipid; D, glycolipid; E, phosphatidyl glycolipid; F, lipoteichoic acid.) (From Van Driel D et al: Cellular location of the lipoteichoic acids of Lactobacillus fermenti NCTC 6991 and Lactobacillus casei NCTC 6375. J Ultrastruct Res 1971;43:483.)



The teichuronic acids are similar polymers, but the repeat units include sugar acids (such as N-acetylmannosuronic or D-glucosuronic acid) instead of phosphoric acids. They are synthesized in place of teichoic acids when phosphate is limiting.

Polysaccharides

The hydrolysis of gram-positive walls has yielded, from certain species, neutral sugars such as mannose, arabinose, rhamnose, and glucosamine and acidic sugars such as glucuronic acid and mannuronic acid. It has been proposed that these sugars exist as subunits of polysaccharides in the cell wall; the discovery, however, that teichoic and teichuronic acids may contain a variety of sugars (Figure 2–16A) leaves the true origin of these sugars uncertain.

Special Components of Gram-Negative Cell Walls

Gram-negative cell walls contain three components that lie outside of the peptidoglycan layer: lipoprotein, outer membrane, and lipopolysaccharide (Figure 2–17).

Figure 2–17.




Molecular representation of the envelope of a gram-negative bacterium. Ovals and rectangles represent sugar residues, whereas circles depict the polar head groups of the glycerophospholipids (phosphatidylethanolamine and phosphatidylglycerol). (MDO, membrane-derived oligosaccharides.) The core region shown is that of E coli K-12, a strain that does not normally contain an O-antigen repeat unless transformed with an appropriate plasmid.

(Reproduced, with permission, from Raetz CRH: Bacterial endotoxins: Extraordinary lipids that activate eucaryotic signal transduction. J Bacteriol 1993;175:5745.)



Outer Membrane

The outer membrane is chemically distinct from all other biological membranes. It is a bilayered structure; its inner leaflet resembles in composition that of the cell membrane while its outer leaflet contains a distinctive component, a lipopolysaccharide (LPS) (see below). As a result, the leaflets of this membrane are asymmetrical, and the properties of this bilayer differ considerably from those of a symmetrical biologic membrane such as the cell membrane.

The ability of the outer membrane to exclude hydrophobic molecules is an unusual feature among biologic membranes and serves to protect the cell (in the case of enteric bacteria) from deleterious substances such as bile salts. Because of its lipid nature, the outer membrane would be expected to exclude hydrophilic molecules as well. However, the outer membrane has special channels, consisting of protein molecules called porins, that permit the passive diffusion of low-molecular-weight hydrophilic compounds like sugars, amino acids, and certain ions. Large antibiotic molecules penetrate the outer membrane relatively slowly, which accounts for the relatively high antibiotic resistance of gram-negative bacteria. The permeability of the outer membrane varies widely from one gram-negative species to another; in Pseudomonas aeruginosa, for example, which is extremely resistant to antibacterial agents, the outer membrane is 100 times less permeable than that of E coli.

The major proteins of the outer membrane, named according to the genes that code for them, have been placed into several functional categories on the basis of mutants in which they are lacking and on the basis of experiments in which purified proteins have been reconstituted into artificial membranes. Porins, exemplified by OmpC, D, and F and PhoE of E coli and Salmonella Typhimurium, are trimeric proteins that penetrate both faces of the outer membrane (Figure 2–18). They form relatively nonspecific pores that permit the free diffusion of small hydrophilic solutes across the membrane. The porins of different species have different exclusion limits, ranging from molecular weights of about 600 in E coli and S typhimurium to more than 3000 in P aeruginosa.

Figure 2–18.






A: General fold of a porin monomer (OmpF porin from E coli). The large hollow -barrel structure is formed by antiparallel arrangement of 16 -strands. The strands are connected by short loops or regular turns on the periplasmic rim (bottom), whereas long irregular loops face the cell exterior (top). The internal loop, which connects -strands 5 and 6 and extends inside the barrel, is highlighted in dark. The chain terminals are marked. The surface closest to the viewer is involved in subunit contacts. B: Schematic representation of the OmpF trimer. The view is from the extracellular space along the molecular threefold symmetry axis.

(Reproduced, with permission, from Schirmer T: General and specific porins from bacterial outer membranes. J Struct Biol 1998;121:101.)



Members of a second group of outer membrane proteins, which resemble porins in many ways, are exemplified by LamB and Tsx. LamB, an inducible porin that is also the receptor for lambda bacteriophage, is responsible for most of the transmembrane diffusion of maltose and maltodextrins; Tsx, the receptor for T6 bacteriophage, is responsible for the transmembrane diffusion of nucleosides and some amino acids. LamB allows some passage of other solutes; however, its relative specificity may reflect weak interactions of solutes with configuration-specific sites within the channel.

The OmpA protein is an abundant protein in the outer membrane. The OmpA protein participates in the anchoring of the outer membrane to the peptidoglycan layer; it is also the sex pilus receptor in F-mediated bacterial conjugation (Chapter 7).

The outer membrane also contains a set of less abundant proteins that are involved in the transport of specific molecules such as vitamin B12 and iron-siderophore complexes. They show high affinity for their substrates and probably function like the classic carrier transport systems of the cytoplasmic membrane. The proper function of these proteins requires energy coupled through a protein called TonB. Additional minor proteins include a limited number of enzymes, among them phospholipases and proteases.

The topology of the major proteins of the outer membrane, based on cross-linking studies and analyses of functional relationships, is shown in Figure 2–17. The outer membrane is connected to both the peptidoglycan layer and the cytoplasmic membrane. The connection with the peptidoglycan layer is primarily mediated by the outer membrane lipoprotein (see below). About one-third of the lipoprotein molecules are covalently linked to peptidoglycan and help hold the two structures together. A noncovalent association of some of the porins with the peptidoglycan layer plays a lesser role in connecting the outer membrane with this structure. Outer membrane proteins are synthesized on ribosomes bound to the cytoplasmic surface of the cell membrane; how they are transferred to the outer membrane is still uncertain, but one hypothesis suggests that transfer occurs at zones of adhesion between the cytoplasmic and outer membranes, which are visible in the electron microscope. Unfortunately, firm evidence for such areas of adhesion has proven hard to come by.

Lipopolysaccharide (LPS)

The LPS of gram-negative cell walls consists of a complex glycolipid, called lipid A, to which is attached a polysaccharide made up of a core and a terminal series of repeat units (Figure 2–19A). The lipid A component is embedded in the outer leaflet of the membrane anchoring the LPS. LPS is synthesized on the cytoplasmic membrane and transported to its final exterior position. The presence of LPS is required for the function of many outer membrane proteins.

Figure 2–19.




The lipopolysaccharide (LPS) of the gram-negative cell envelope. A: A segment of the polymer, showing the arrangements of the major constituents. B: The structure of lipid A of Salmonella Typhimurium.C: The polysaccharide core. D: A typical repeat unit (Salmonella Newington). Serologic specificity is determined in part by the type of bond ( or ) between monosaccharide units.



Lipid A consists of phosphorylated glucosamine disaccharide units to which are attached a number of long-chain fatty acids (Figure 2–19B). -Hydroxymyristic acid, a C14 fatty acid, is always present and is unique to this lipid; the other fatty acids, along with substituent groups on the phosphates, vary according to the bacterial species.

The polysaccharide core, shown in Figure 2–19C, is similar in all gram-negative species that have LPS and includes two characteristic sugars, ketodeoxyoctanoic acid (KDO) and a heptose. Each species, however, contains a unique repeat unit, that of Salmonella Newington being shown in Figure 2–19D. The repeat units are usually linear trisaccharides or branched tetra- or pentasaccharides. The repeat unit is referred to as the O antigen. The hydrophilic carbohydrate chains of the O antigen cover the bacterial surface and exclude hydrophobic compounds.

The negatively charged LPS molecules are noncovalently cross-bridged by divalent cations (ie, Ca2+ and Mg2+); this stabilizes the membrane and provides a barrier to hydrophobic molecules. Removal of the divalent cations with chelating agents or their displacement by polycationic antibiotics such as polymyxins and aminoglycosides renders the outer membrane permeable to large hydrophobic molecules.

LPS, which is extremely toxic to animals, has been called the endotoxin of gram-negative bacteria because it is firmly bound to the cell surface and is released only when the cells are lysed. When LPS is split into lipid A and polysaccharide, all of the toxicity is associated with the former. The O antigen is highly immunogenic in a vertebrate animal. Antigenic specificity is conferred by the O antigen as this antigen is highly variable among species and even in strains within a species. The number of possible antigenic types is very great: Over 1000 have been recognized in salmonella alone.

Not all gram-negative bacteria have outer membrane LPS composed of a variable number of repeated oligosaccharide units (see Figure 2–19); the outer membrane glycolipids of bacteria that colonize mucosal surfaces (eg, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, and Haemophilus ducreyi) have relatively short, multiantennary (ie, branched) glycans. These smaller glycolipids have been compared with the "R-type" truncated LPS structures, which lack O-antigens and are produced by rough mutants of enteric bacteria such as E coli. However, their structures more closely resemble those of the glycosphingolipids of mammalian cell membranes, and they are more properly termed lipooligosaccharides (LOS). These molecules exhibit extensive antigenic and structural diversity even within a single strain. LOS is an important virulence factor. Epitopes have been identified on LOS which mimic host structures and may enable these organisms to evade the immune response of the host. Some LOS (eg, those from N gonorrhoeae, N meningitidis, and H ducreyi) have a terminal N-acetyllactosamine (Gal4-GlcNAc) residue which is immunochemically similar to the precursor of the human erythrocyte i antigen. In the presence of a bacterial enzyme called sialyltransferase and a host or bacterial substrate (cytidine monophospho-N-acetylneuraminic acid, CMP-NANA), the N-acetyllactosamine residue is sialylated. This sialylation, which occurs in vivo, provides the organism with the environmental advantages of molecular mimicry of a host antigen and the biologic masking thought to be provided by sialic acids.

Lipoprotein

Molecules of an unusual lipoprotein cross-link the outer membrane and peptidoglycan layers (Figure 2–17). The lipoprotein contains 57 amino acids, representing repeats of a 15-amino-acid sequence; it is peptide-linked to DAP residues of the peptidoglycan tetrapeptide side chains. The lipid component, consisting of a diglyceride thioether linked to a terminal cysteine, is noncovalently inserted in the outer membrane. Lipoprotein is numerically the most abundant protein of gram-negative cells (ca 700,000 molecules per cell). Its function (inferred from the behavior of mutants that lack it) is to stabilize the outer membrane and anchor it to the peptidoglycan layer.

The Periplasmic Space

The space between the inner and outer membranes, called the periplasmic space, contains the peptidoglycan layer and a gel-like solution of proteins. The periplasmic space is approximately 20–40% of the cell volume, which is far from insignificant. The periplasmic proteins include binding proteins for specific substrates (eg, amino acids, sugars, vitamins, and ions), hydrolytic enzymes (eg, alkaline phosphatase and 5'-nucleotidase) that break down nontransportable substrates into transportable ones, and detoxifying enzymes (eg, -lactamase and aminoglycoside-phosphorylase) that inactivate certain antibiotics. The periplasm also contains high concentrations of highly branched polymers of D-glucose, eight to ten residues long, which are variously substituted with glycerol phosphate and phosphatidylethanolamine residues; some contain O-succinyl esters. These so-called membrane-derived oligosaccharides appear to play a role in osmoregulation, since cells grown in media of low osmolarity increase their synthesis of these compounds 16-fold.

The Acid-Fast Cell Wall

Some bacteria, notably the tubercle bacillus (Mycobacterium tuberculosis) and its relatives have cell walls that contain large amounts of waxes, complex branched hydrocarbons (70 to 90 carbons long) known as mycolic acids. The cell wall is composed of peptidoglycan and an external asymmetric lipid bilayer; the inner leaflet contains mycolic acids linked to an arabinoglycan and the outer leaflet contains other extractable lipids. This is a highly ordered lipid bilayer in which proteins are embedded forming water-filled pores through which nutrients and certain drugs can pass slowly. Some compounds can also penetrate the lipid domains of the cell wall albeit slowly. This hydrophobic structure renders these bacteria resistant to many harsh chemicals including detergents and strong acids. If a dye is introduced into these cells by brief heating or treatment with detergents, it cannot be removed by dilute hydrochloric acid, as in other bacteria. These organisms are therefore called acid-fast. The permeability of the cell wall to hydrophilic molecules is 100- to 1000-fold lower than for E coli and may be responsible for the slow growth rate of mycobacteria.

Cell Walls of the Archaea

The Archaea do not have cell walls like the Bacteria. Some have a simple S-layer (see below) often comprised of glycoproteins. Some Archaea have a rigid cell wall composed of polysaccharides or a peptidoglycan called pseudomurein. The pseudomurein differs from the peptidoglycan of bacteria by having L-amino acids rather than D-amino acids and disaccharide units with an -1,3 rather than a -1,4 linkage. Archaea that have a pseudomurein cell wall are gram positive.

Crystalline Surface Layers

Many bacteria, both gram-positive and gram-negative bacteria as well as archaebacteria, possess a two-dimensional crystalline, subunit-type layer lattice of protein or glycoprotein molecules (S-layer) as the outermost component of the cell envelope. In both gram-positive and gram-negative bacteria, this structure is sometimes several molecules thick. In some Archaea, they are the only layer external to the cell membrane.

S-layers are generally composed of a single kind of protein molecule, sometimes with carbohydrates attached. The isolated molecules are capable of self-assembly, ie, they make sheets similar or identical to those present on the cells. S-layer proteins are resistant to proteolytic enzymes and protein-denaturing agents. The function of the S-layer is uncertain but is probably protective. In some cases, it has been shown to protect the cell from wall-degrading enzymes, from invasion by Bdellovibrio bacteriovorous (a predatory bacterium), and from bacteriophages. It also plays a role in the maintenance of cell shape in some species of archaebacteria, and it may be involved in cell adhesion to host epidermal surfaces.

Enzymes that Attack Cell Walls

The 14 linkage of the peptidoglycan backbone is hydrolyzed by the enzyme lysozyme, which is found in animal secretions (tears, saliva, nasal secretions) as well as in egg white. Gram-positive bacteria treated with lysozyme in low-osmotic-strength media lyse; if the osmotic strength of the medium is raised to balance the internal osmotic pressure of the cell, free spherical bodies called protoplasts are liberated. The outer membrane of the gram-negative cell wall prevents access of lysozyme unless disrupted by an agent such as ethylenediaminetetraacetic acid (EDTA), a compound that chelates divalent cations; in osmotically protected media, cells treated with EDTA-lysozyme form spheroplasts that still possess remnants of the complex gram-negative wall, including the outer membrane.

Bacteria themselves possess a number of autolysins, hydrolytic enzymes that attack peptidoglycan, including muramidases, glucosaminidases, endopeptidases, and carboxypeptidases. These enzymes catalyze the turnover or degradation of peptidoglycan in bacteria. These enzymes presumably participate in cell wall growth and turnover and in cell separation, but their activity is most apparent during the dissolution of dead cells (autolysis).

Enzymes that degrade bacterial cell walls are also found in cells that digest whole bacteria, eg, protozoa and the phagocytic cells of higher animals.

Cell Wall Growth

Cell wall synthesis is necessary for cell division; however, the incorporation of new cell wall material varies with the shape of the bacterium. Rod-shaped bacteria (eg, E coli, Bacillus subtilis) have two modes of cell wall synthesis; new peptidoglycan is inserted along a helical path leading to elongation of the cell, and is inserted in a closing ring around the future division site, leading to the formation of the division septum. Coccoid cells such as S aureus do not seem to have an elongation mode of cell wall synthesis. Instead, new peptidoglycan is inserted only at the division site. A third form of cell wall growth is exemplified by S pneumoniae, which are not true cocci, as their shape is not totally round, but instead have the shape of a rugby ball. S pneumoniae synthesize cell wall not only at the septum but also at the so-called "equatorial rings" (Figure 2–20).

Figure 2–20.




Incorporation of new cell wall in differently shaped bacteria. Rod-shaped bacteria such as Bacillus subtilis or Escherichia coli have two modes of cell wall synthesis: New peptidoglycan is inserted along a helical path (A), leading to elongation of the lateral wall, and is inserted in a closing ring around the future division site, leading to the formation of the division septum (B). Streptococcus pneumoniae cells have the shape of a rugby ball and elongate by inserting new cell wall material at the so-called equatorial rings (A), which correspond to an outgrowth of the cell wall that encircles the cell. An initial ring is duplicated, and the two resultant rings are progressively separated, marking the future division sites of the daughter cells. The division septum is then synthesized in the middle of the cell (B). Round cells such as Staphylococcus aureus do not seem to have an elongation mode of cell wall synthesis. Instead, new peptidoglycan is inserted only at the division septum (B). Elongation-associated growth is indicated in the lighter color, and division-associated growth is indicated in the darker color.

(Reproduced, with permission, from Scheffers DJ and Pinho MG: Microbiol Mol Biol Rev 2005;69:585.)



Protoplasts, Spheroplasts, and L Forms

Removal of the bacterial wall may be accomplished by hydrolysis with lysozyme or by blocking peptidoglycan synthesis with an antibiotic such as penicillin. In osmotically protective media, such treatments liberate protoplasts from gram-positive cells and spheroplasts (which retain outer membrane and entrapped peptidoglycan) from gram-negative cells.

If such cells are able to grow and divide, they are called L forms. L forms are difficult to cultivate and usually require a medium that is solidified with agar as well as having the right osmotic strength. L forms are produced more readily with penicillin than with lysozyme, suggesting the need for residual peptidoglycan.

Some L forms can revert to the normal bacillary form upon removal of the inducing stimulus. Thus, they are able to resume normal cell wall synthesis. Others are stable and never revert. The factor that determines their capacity to revert may again be the presence of residual peptidoglycan, which normally acts as a primer in its own biosynthesis.

Some bacterial species produce L forms spontaneously. The spontaneous or antibiotic-induced formation of L forms in the host may produce chronic infections, the organisms persisting by becoming sequestered in protective regions of the body. Since L-form infections are relatively resistant to antibiotic treatment, they present special problems in chemotherapy. Their reversion to the bacillary form can produce relapses of the overt infection.

The Mycoplasmas

The mycoplasmas are cell wall-lacking bacteria containing no peptidoglycan. There are also wall-less Archaea, but they have been less well studied. Genomic analysis places the mycoplasmas close to the gram-positive bacteria from which they may have been derived. Mycoplasmas lack a target for cell wall-inhibiting antimicrobial agents (eg, penicillins and cephalosporins) and are therefore resistant to these drugs. Some, like Mycoplasma pneumoniae, an agent of pneumonia, contain sterols in their membranes. The difference between L forms and mycoplasmas is that when the murein is allowed to reform, L forms revert to their original bacteria shape, but mycoplasmas never do.

Capsule & Glycocalyx

Many bacteria synthesize large amounts of extracellular polymer when growing in their natural environments. With one known exception (the poly-D-glutamic acid capsules of Bacillus anthracis and Bacillus licheniformis), the extracellular material is polysaccharide (Table 2–1). The terms capsule and slime layer are frequently used to describe polysaccharide layers; the more inclusive term glycocalyx is also used. Glycocalyx is defined as the polysaccharide-containing material lying outside the cell. A condensed, well-defined layer closely surrounding the cell that excludes particles, such as India ink, is referred to as a capsule (Figure 2–21). If the glycocalyx is loosely associated with the cell and does not exclude particles, it is referred to as a slime layer. Extracellular polymer is synthesized by enzymes located at the surface of the bacterial cell. Streptococcus mutans, for example, uses two enzymes—glucosyl transferase and fructosyl transferase—to synthesize long-chain dextrans (poly-D-glucose) and levans (poly-D-fructose) from sucrose. These polymers are called homopolymers. Polymers containing more than one kind of monosaccharide are called heteropolymers.

Table 2–1. Chemical Composition of the Extracellular Polymer in Selected Bacteria.



Organism Polymer Chemical Subunits
Bacillus anthracis Polypeptide D-Glutamic acid
Enterobacter aerogenes Complex polysaccharide Glucose, fucose, glucuronic acid
Neisseria meningitidis Homopolymers and heteropolymers, eg,
Serogroup A Partially O-acetylated N-acetylmannosaminephosphate
Serogroup B N-Acetylneuraminic acid (sialic acid)
Serogroup C Acetylated sialic acid
Serogroup 135 Galactose, sialic acid
Streptococcus pneumoniae (pneumococcus) Complex polysaccharide (many types), eg,
Type II Rhamnose, glucose, glucuronic acid
Type III Glucose, glucuronic acid
Type VI Galactose, glucose, rhamnose
Type XIV Galactose, glucose, N-acetylglucosamine
Type XVIII Rhamnose, glucose
Streptococcus pyogenes (group A) Hyaluronic acid N-Acetylglucosamine, glucuronic acid
Streptococcus salivarius Levan Fructose



Figure 2–21.






Bacterial capsules. A: Bacillus anthracis M’Faydean capsule stain, grown at 35 °C, in defibrinated horse blood. B: Demonstration of the presence of a capsule in Bacillus anthracis by negative staining with India ink. This method is useful for improving visualization of encapsulated bacteria in clinical samples such as blood, blood culture bottles, or cerebrospinal fluid. (CDC, courtesy of Larry Stauffer, Oregon State Public Health Laboratory.)



The capsule contributes to the invasiveness of pathogenic bacteria—encapsulated cells are protected from phagocytosis unless they are coated with anticapsular antibody. The glycocalyx plays a role in the adherence of bacteria to surfaces in their environment, including the cells of plant and animal hosts. S mutans, for example, owes its capacity to adhere tightly to tooth enamel to its glycocalyx. Bacterial cells of the same or different species become entrapped in the glycocalyx, which forms the layer known as plaque on the tooth surface; acidic products excreted by these bacteria cause dental caries (Chapter 11). The essential role of the glycocalyx in this process—and its formation from sucrose—explains the correlation of dental caries with sucrose consumption by the human population. Because outer polysaccharide layers bind a significant amount of water, the glycocalyx layer may also play a role in resistance to desiccation.

Flagella

Structure

Bacterial flagella are thread-like appendages composed entirely of protein, 12–30 nm in diameter. They are the organs of locomotion for the forms that possess them. Three types of arrangement are known: monotrichous (single polar flagellum), lophotrichous (multiple polar flagella), and peritrichous (flagella distributed over the entire cell). The three types are illustrated in Figure 2–22.

A bacterial flagellum is made up of several thousand molecules of a protein subunit called flagellin. In a few organisms (eg, caulobacter), flagella are composed of two types of flagellin, but in most only a single type is found. The flagellum is formed by the aggregation of subunits to form a helical structure. If flagella are removed by mechanically agitating a suspension of bacteria, new flagella are rapidly formed by the synthesis, aggregation, and extrusion of flagellin subunits; motility is restored within 3–6 minutes. The flagellins of different bacterial species presumably differ from one another in primary structure. They are highly antigenic (H antigens), and some of the immune responses to infection are directed against these proteins.

The flagellum is attached to the bacterial cell body by a complex structure consisting of a hook and a basal body. The hook is a short curved structure that appears to act as the universal joint between the motor in the basal structure and the flagellum. The basal body bears a set of rings, one pair in gram-positive bacteria and two pairs in gram-negative bacteria. An electron micrograph and interpretative diagrams of the gram-negative structure are shown in Figures 2–23 and 2–24; the rings labeled L and P are absent in gram-positive cells. The complexity of the bacterial flagellum is revealed by genetic studies, which show that over 40 gene products are involved in its assembly and function.

Figure 2–23.




Electron micrograph of a negatively stained lysate of Rhodospirillum molischianum, showing the basal structure of an isolated flagellum.

(Reproduced, with permission, from Cohen-Bazire G, London L: Basal organelles of bacterial flagella. J Bacteriol 1967;94:458.)



Figure 2–24.




A: General structure of the flagellum of a gram-negative bacterium, such as E coli or S Typhimurium. The filament-hook-basal body complex has been isolated and extensively characterized. The location of the export apparatus has not been demonstrated. B: An exploded diagram of the flagellum showing the substructures and the proteins from which they are constructed. The FliF protein is responsible for the M-ring feature, S-ring feature, and collar feature of the substructure shown, which is collectively termed the MS ring. The location of FliE with respect to the MS ring and the rod—and the order of the FlgB, FlgC, and FlgF proteins within the proximal rod is not known.

(From Macnab RM: Genetics and biogenesis of bacterial flagella. Annu Rev Genet 1992;26:131. Reproduced with permission from Annual Review of Genetics, Volume 26, © 1992 by Annual Reviews.)



Flagella are made stepwise (Figure 2–24). First the basal body is assembled and inserted into the cell envelope. Then the hook is added, and finally, the filament is assembled progressively by the addition of flagellin subunits to its growing tip. The flagellin subunits are extruded through a hollow central channel in the flagella; when it reaches the tip it condenses with its predecessors, and thus the filament elongates.

Motility

Bacterial flagella are semirigid helical rotors to which the cell imparts a spinning movement. Rotation is driven by the flow of protons into the cell down the gradient produced by the primary proton pump (see above); in the absence of a metabolic energy source, it can be driven by a proton motive force generated by ionophores. Bacteria living in alkaline environments (alkalophiles) use the energy of the sodium ion gradient—rather than the proton gradient—to drive the flagellar motor (Figure 2–25).

Figure 2–25.




Structural components within the basal body of the flagellum allow the inner portion of this structure, the rods of the basal body, and the attached hook-filament complex to rotate. The outer rings remain statically in contact with the inner and outer cell membranes and cell wall (murein), anchoring the flagellum complex to the bacterial cell envelope. Rotation is driven by the flow of protons through the motor from the periplasmic space, outside the cell membrane, into the cytoplasm in response to the electric field and proton gradient across the membrane, which together comprise the proton motive force. A switch determines the direction of rotation, which in turn determines whether the bacteria swim forward (due to counterclockwise rotation of the flagellum) or tumble (due to clockwise rotation of the flagellum).

(Reproduced, with permission, from Saier MH Jr: Peter Mitchell and his chemiosmotic theories. ASM News 1997;63:13.)



All of the components of the flagellar motor are located in the cell envelope. Flagella attached to isolated, sealed cell envelopes rotate normally when the medium contains a suitable substrate for respiration or when a proton gradient is artificially established.

When a peritrichous bacterium swims, its flagella associate to form a posterior bundle that drives the cell forward in a straight line by counterclockwise rotation. At intervals, the flagella reverse their direction of rotation and momentarily dissociate, causing the cell to tumble until swimming resumes in a new, randomly determined direction. This behavior makes possible the property of chemotaxis: A cell that is moving away from the source of a chemical attractant tumbles and reorients itself more frequently than one that is moving toward the attractant, the result being the net movement of the cell toward the source. The presence of a chemical attractant (such as a sugar or an amino acid) is sensed by specific receptors located in the cell membrane (in many cases, the same receptor also participates in membrane transport of that molecule). The bacterial cell is too small to be able to detect the existence of a spatial chemical gradient (ie, a gradient between its two poles); rather, experiments show that it detects temporal gradients, ie, concentrations that decrease with time during which the cell is moving away from the attractant source and increase with time during which the cell is moving toward it.

Some compounds act as repellants rather than attractants. One mechanism by which cells respond to attractants and repellents involves a cGMP-mediated methylation and demethylation of specific proteins in the membrane. Attractants cause a transient inhibition of demethylation of these proteins, while repellents stimulate their demethylation.

The mechanism by which a change in cell behavior is brought about in response to a change in the environment is called sensory transduction. Sensory transduction is responsible not only for chemotaxis but also for aerotaxis (movement toward the optimal oxygen concentration), phototaxis (movement of photosynthetic bacteria toward the light), and electron acceptor taxis (movement of respiratory bacteria toward alternative electron acceptors, such as nitrate and fumarate). In these three responses, as in chemotaxis, net movement is determined by regulation of the tumbling response.

Pili (Fimbriae)

Many gram-negative bacteria possess rigid surface appendages called pili (L "hairs") or fimbriae (L "fringes"). They are shorter and finer than flagella; like flagella, they are composed of structural protein subunits termed pilins. Some pili contain a single type of pilin, others more than one. Minor proteins termed adhesins are located at the tips of pili and are responsible for the attachment properties. Two classes can be distinguished: ordinary pili, which play a role in the adherence of symbiotic and pathogenic bacteria to host cells, and sex pili, which are responsible for the attachment of donor and recipient cells in bacterial conjugation (see Chapter 7). Pili are illustrated in Figure 2–26, in which the sex pili have been coated with phage particles for which they serve as specific receptors.

Figure 2–26.




Surface appendages of bacteria. Electron micrograph of a cell of E coli possessing three types of appendages: ordinary pili (short, straight bristles), a sex pilus (longer, flexible, with phage particles attached), and several flagella (longest, thickest). Diameters: ordinary pili: 7 nm; sex pili: 8.5 nm; flagella: 25 nm.

(Courtesy of J Carnahan and C Brinton.)



Motility via pili is completely different from flagellar motion. Pilin molecules are arranged helically to form a straight cylinder that does not rotate and lacks a complete basal body. Their tips strongly adhere to surfaces at a distance from the cells. Pili then depolymerize from the inner end, thus retracting inside the cell. The result is that the bacterium moves in the direction of the adhering tip. This kind of surface motility is called twitching and is widespread among piliated bacteria. Unlike flagella, pili grow from the inside of the cell outward.

The virulence of certain pathogenic bacteria depends on the production not only of toxins but also of "colonization antigens," which are ordinary pili that provide the cells with adherent properties. In enteropathogenic E coli strains, both the enterotoxins and the colonization antigens (pili) are genetically determined by transmissible plasmids, as discussed in Chapter 7.

In one group of gram-positive cocci, the streptococci, fimbriae are the site of the main surface antigen, the M protein. Lipoteichoic acid, associated with these fimbriae, is responsible for the adherence of group A streptococci to epithelial cells of their hosts.

Pili of different bacteria are antigenically distinct and elicit the formation of antibodies by the host. Antibodies against the pili of one bacterial species will not prevent the attachment of another species. Some bacteria (see Chapter 21), such as N gonorrhoeae, are able to make pili of different antigenic types (antigenic variation) and thus can still adhere to cells in the presence of antibodies to their original type of pili. Like capsules, pili inhibit the phagocytic ability of leukocytes.

Endospores

Members of several bacterial genera are capable of forming endospores (see Figure 2–27). The two most common are gram-positive rods: the obligately aerobic genus Bacillus and the obligately anaerobic genus Clostridium. The other bacteria known to form endospores are Thermoactinomyces, Sporolactobacillus, Sporosarcina, Sporotomaculum, Sporomusa, and Sporohalobacter. These organisms undergo a cycle of differentiation in response to environmental conditions: The process, sporulation, is triggered by near depletion of any of several nutrients (carbon, nitrogen, or phosphorous). Each cell forms a single internal spore that is liberated when the mother cell undergoes autolysis. The spore is a resting cell, highly resistant to desiccation, heat, and chemical agents; when returned to favorable nutritional conditions and activated (see below), the spore germinates to produce a single vegetative cell.

Figure 2–27.




Sporulating cells of bacillus species. A: Unidentified bacillus from soil. B: B cereus. C: B megaterium.

(Reproduced, with permission, from Robinow CF, in: Structure. Vol 1 of: The Bacteria: A Treatise on Structure and Function. Gunsalus IC, Stanier RY [editors]. Academic Press, 1960.)



Sporulation

The sporulation process begins when nutritional conditions become unfavorable, near depletion of the nitrogen or carbon source (or both) being the most significant factor. Sporulation occurs massively in cultures that have terminated exponential growth as a result of this near depletion.

Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components. These changes represent a true process of differentiation: A series of genes whose products determine the formation and final composition of the spore are activated. These changes involve alterations in the transcriptional specificity of RNA polymerase, which is determined by the association of the polymerase core protein with one or another promoter-specific protein called a sigma factor. During vegetative growth, a sigma factor designated A predominates. Then, during sporulation, five other sigma factors are formed that cause various spore genes to be expressed at various times in specific locations.

The sequence of events in sporulation is highly complex: Differentiation of a vegetative cell of B subtilis into an endospore takes about 7 hours under laboratory conditions. Different morphologic and chemical events occur at sequential stages of the process. Seven different stages have been identified.

Morphologically, sporulation begins with the formation of an axial filament (Figure 2–28). The process continues with an infolding of the membrane so as to produce a double membrane structure whose facing surfaces correspond to the cell wall-synthesizing surface of the cell envelope. The growing points move progressively toward the pole of the cell so as to engulf the developing spore.

Figure 2–28.




The stages of endospore formation.

(Reproduced, with permission, from Merrick MJ: Streptomyces. In: Developmental Biology of Procaryotes. Parish JH [editor]. Univ California Press, 1979.)



The two spore membranes now engage in the active synthesis of special layers that will form the cell envelope: the spore wall and the cortex, lying outside the facing membranes. In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a set of unique spore constituents.

Properties of Endospores

Core

The core is the spore protoplast. It contains a complete nucleus (chromosome), all of the components of the protein-synthesizing apparatus, and an energy-generating system based on glycolysis. Cytochromes are lacking even in aerobic species, the spores of which rely on a shortened electron transport pathway involving flavoproteins. A number of vegetative cell enzymes are increased in amount (eg, alanine racemase), and a number of unique enzymes are formed (eg, dipicolinic acid synthetase). Spores contain no reduced pyridine nucleotides or ATP. The energy for germination is stored as 3-phosphoglycerate rather than as ATP.

The heat resistance of spores is due in part to their dehydrated state and in part to the presence in the core of large amounts (5–15% of the spore dry weight) of calcium dipicolinate, which is formed from an intermediate of the lysine biosynthetic pathway (see Figure 6–18). In some way not yet understood, these properties result in the stabilization of the spore enzymes, most of which exhibit normal heat lability when isolated in soluble form.

Spore Wall

The innermost layer surrounding the inner spore membrane is called the spore wall. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.

Cortex

The cortex is the thickest layer of the spore envelope. It contains an unusual type of peptidoglycan, with many fewer cross-links than are found in cell wall peptidoglycan. Cortex peptidoglycan is extremely sensitive to lysozyme, and its autolysis plays a role in spore germination.

Coat

The coat is composed of a keratin-like protein containing many intramolecular disulfide bonds. The impermeability of this layer confers on spores their relative resistance to antibacterial chemical agents.

Exosporium

The exosporium is a lipoprotein membrane containing some carbohydrate.

Germination

The germination process occurs in three stages: activation, initiation, and outgrowth.

Activation

Most endospores cannot germinate immediately after they have formed. But they can germinate after they have rested for several days or are first activated, in a nutritionally rich medium, by one or another agent that damages the spore coat. Among the agents that can overcome spore dormancy are heat, abrasion, acidity, and compounds containing free sulfhydryl groups.

Initiation

Once activated, a spore will initiate germination if the environmental conditions are favorable. Different species have evolved receptors that recognize different effectors as signaling a rich medium: Thus, initiation is triggered by L-alanine in one species and by adenosine in another. Binding of the effector activates an autolysin that rapidly degrades the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes.

Outgrowth

Degradation of the cortex and outer layers results in the emergence of a new vegetative cell consisting of the spore protoplast with its surrounding wall. A period of active biosynthesis follows; this period, which terminates in cell division, is called outgrowth. Outgrowth requires a supply of all nutrients essential for cell growth.

Staining

Stains combine chemically with the bacterial protoplasm; if the cell is not already dead, the staining process itself will kill it. The process is thus a drastic one and may produce artifacts.

The commonly used stains are salts. Basic stains consist of a colored cation with a colorless anion (eg, methylene blue+ chloride–); acidic stains are the reverse (eg, sodium+ eosinate–). Bacterial cells are rich in nucleic acid, bearing negative charges as phosphate groups. These combine with the positively charged basic dyes. Acidic dyes do not stain bacterial cells and hence can be used to stain background material a contrasting color (see Negative Staining, below).

The basic dyes stain bacterial cells uniformly unless the cytoplasmic RNA is destroyed first. Special staining techniques can be used, however, to differentiate flagella, capsules, cell walls, cell membranes, granules, nucleoids, and spores.

The Gram Stain

An important taxonomic characteristic of bacteria is their response to Gram stain. The Gram-staining property appears to be a fundamental one, since the Gram reaction is correlated with many other morphologic properties in phylogenetically related forms (Chapter 3). An organism that is potentially gram-positive may appear so only under a particular set of environmental conditions and in a young culture.

The Gram-staining procedure (see Chapter 47 for details) begins with the application of a basic dye, crystal violet. A solution of iodine is then applied; all bacteria will be stained blue at this point in the procedure. The cells are then treated with alcohol. Gram-positive cells retain the crystal violet-iodine complex, remaining blue; gram-negative cells are completely decolorized by alcohol. As a last step, a counterstain (such as the red dye safranin) is applied so that the decolorized gram-negative cells will take on a contrasting color; the gram-positive cells now appear purple.

The basis of the differential Gram reaction is the structure of the cell wall, as discussed earlier in this chapter.

The Acid-Fast Stain

Acid-fast bacteria are those that retain carbolfuchsin (basic fuchsin dissolved in a phenol-alcohol-water mixture) even when decolorized with hydrochloric acid in alcohol. A smear of cells on a slide is flooded with carbolfuchsin and heated on a steam bath. Following this, the discolorization with acid-alcohol is carried out, and finally a contrasting (blue or green) counterstain is applied (see Chapter 47). Acid-fast bacteria (mycobacteria and some of the related actinomycetes) appear red; others take on the color of the counterstain.

Negative Staining

This procedure involves staining the background with an acidic dye, leaving the cells contrastingly colorless. The black dye nigrosin is commonly used. This method is used for those cells or structures difficult to stain directly (Figure 2–21B).

The Flagella Stain

Flagella are too fine (12–30 nm in diameter) to be visible in the light microscope. However, their presence and arrangement can be demonstrated by treating the cells with an unstable colloidal suspension of tannic acid salts, causing a heavy precipitate to form on the cell walls and flagella. In this manner, the apparent diameter of the flagella is increased to such an extent that subsequent staining with basic fuchsin makes the flagella visible in the light microscope. Figure 2–29 shows cells stained by this method.

Figure 2–29.




Flagella stain of pseudomonas species.

(Courtesy of Leifson E: J Bacteriol 1951;62:377.)



In peritrichous bacteria, the flagella form into bundles during movement, and such bundles may be thick enough to be observed on living cells by darkfield or phase contrast microscopy.

The Capsule Stain

Capsules are usually demonstrated by the negative staining procedure or a modification of it (Figure 2–21). One such "capsule stain" (Welch method) involves treatment with hot crystal violet solution followed by a rinsing with copper sulfate solution. The latter is used to remove excess stain because the conventional washing with water would dissolve the capsule. The copper salt also gives color to the background, with the result that the cell and background appear dark blue and the capsule a much paler blue.

Staining of Nucleoids

Nucleoids are stainable with the Feulgen stain, which is specific for DNA (see Figure 2–4).

The Spore Stain

Spores are most simply observed as intracellular refractile bodies in unstained cell suspensions or as colorless areas in cells stained by conventional methods (Figure 2–27). The spore wall is relatively impermeable, but dyes can be made to penetrate it by heating the preparation. The same impermeability then serves to prevent decolorization of the spore by a period of alcohol treatment sufficient to decolorize vegetative cells. The latter can finally be counterstained. Spores are commonly stained with malachite green or carbolfuchsin.

Morphologic Changes during Growth

Cell Division

Most bacteria divide by binary fission into two equal progeny cells. In a growing culture of a rod-shaped bacterium such as E coli, cells elongate and then form a partition that eventually separates the cell into two daughter cells. The partition is referred to as a septum and is a result of the inward growth of the cytoplasmic membrane and cell wall from opposing directions until the two daughter cells are pinched off. The chromosomes, which have doubled in number preceding the division, are distributed equally to the two daughter cells.

Although bacteria lack a mitotic spindle, the septum is formed in such a way as to separate the two sister chromosomes formed by chromosomal replication. This is accomplished by the attachment of the chromosome to the cell membrane. According to one model, completion of a cycle of DNA replication triggers active membrane synthesis between the sites of attachment of the two sister chromosomes. The chromosomes are then pushed apart by the inward growth of the septum, one copy going to each daughter cell.

Cell Groupings

If the cells remain temporarily attached following division, certain characteristic groupings result. Depending on the plane of division and the number of divisions through which the cells remain attached, the following may occur in the coccal forms: chains (streptococci), pairs (diplococci), cubical bundles (sarcinae), or flat plates. Rods may form pairs or chains.

Following fission of some bacteria, characteristic post-division movements occur. For example, a "whipping" motion can bring the cells into parallel positions; repeated division and whipping result in the "palisade" arrangement characteristic of diphtheria bacilli.

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