Bacteriology at UW-Madison

The Microbial World

Lectures in Microbiology by Kenneth Todar PhD    University of Wisconsin-Madison    Department of Bacteriology

Structure and Function of Bacterial Cells

Figure 1. Structure of a bacterial endospore, renowned as the most durable and long-lived type of cell on earth. Drawing by Vaike Haas, University of Wisconsin.

Table 1: Macromolecules that make up cell material

The overall chemical composition of a bacterial cell may be inferred by chemical analysis of a bacterium such as E. coli, which is displayed in Table 2. The macromolecules comprise about 96 percent of the dry weight of the cell ("dry weight" is the remaining material after all water is removed). Small molecules and inorganic ions, which are constituents of the cytoplasm, comprise the remaining 4 percent.

Table 2. Molecular composition of E. coli under conditions of balanced growth.

Procaryotic Cell Architecture

At one time it was thought that bacteria were essentially "bags of enzymes" with no inherent cellular architecture. The development of the electron microscope in the 1950s revealed the distinct anatomical features of bacteria and confirmed the suspicion that they lacked a nuclear membrane. Structurally, a bacterial cell (Figure 2 below) has three architectural regions: appendages (attachments to the cell surface) in the form of flagella and pili (or fimbriae); a cell envelope consisting of a capsule, cell wall and plasma membrane; and a cytoplasmic region that contains the cell chromosome (DNA) and ribosomes and various sorts of inclusions. In this lecture, we will discuss the anatomical structures of procaryotic cells in relation to their adaptation, function and behavior in natural environments.



Figure 2.  Schematic drawing of a typical bacterial cell.

Figure 3. Electron micrograph of an ultra-thin section of a dividing pair of group A streptococci (20,000X). The cell surface fibrils, consisting primarily of protein, are evident. The bacterial cell wall, to which the fibrils are attached, is also clearly seen as the light staining region between the fibrils and the dark staining cell interior. Cell division in progress is indicated by the new septum formed between the two cells and by the indentation of the cell wall near the cell equator. The streptococcal cell diameter is equal to approximately one micron. Electron micrograph of Streptococcus pyogenes by Maria Fazio and Vincent A. Fischetti, Ph.D. with permission. The Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University.

Figure 4. Electron micrograph of an ultra-thin section of a dividing pair of enterococci

Table 3.  Characteristics of typical bacterial cell structures.

Appendages

Figure 5. Salmonella enterica.  Salmonella is an enteric bacterium related to E. coli. The enterics are motile by means of peritrichous flagella.

Flagella

Flagella are filamentous protein structures attached to the cell surface that provide the swimming movement for most motile procaryotes. Procaryotic flagella are much thinner than eucaryotic flagella; the diameter of a procaryotic flagellum is about 20 nanometers, well-below the resolving power of the light microscope. The flagellar filament is rotated by a motor apparatus in the plasma membrane allowing the cell to swim in fluid environments.

The ultrastructure of the flagellum of E. coli is illustrated in Figure 5 below (after Dr. Julius Adler of the University of Wisconsin). The flagellar apparatus consists of several distinct proteins: a system of rings imbedded in the cell envelope (the basal body), a hook-like structure near the cell surface, and the flagellar filament. The innermost rings, the M and S rings, located in the plasma membrane, comprise the motor apparatus. As the M ring turns, powered by an influx of protons, the rotary motion is transferred to the filament which rotates thereby propelling the bacterium.

 

Figure 6. The ultrastructure of a bacterial flagellum (after J. Adler). Measurements are in nanometers. The flagellum of E. coli consists of three parts, filament, hook and basal body, all composed of different proteins. The basal body and hook anchor the whip-like filament to the cell surface. The basal body consists of four ring-shaped proteins stacked like donuts around a central rod in the cell envelope. The inner rings, associated with the plasma membrane, are the flagellar powerhouse for activating the filament. The filament rotates and contracts which propels and steers the cell during movement. 

Flagella may be variously distributed over the surface of bacterial cells in distinguishing patterns, but basically flagella are either polar (one or more flagella arising from one or both poles of the cell) or peritrichous (lateral flagella distributed over the entire cell surface). Flagellar distribution is a genetically-distinct trait that is occasionally used to characterize or distinguish bacteria. For example, among Gram-negative rods, pseudomonads have polar flagella to distinguish them from enteric bacteria, which have peritrichous flagella.

Figure 7a. Different arrangements of bacterial flagella. Swimming motility, powered by flagella, occurs in half the bacilli and most of the spirilla. Flagellar arrangements, which can be determined by staining and microscopic observation, may be a clue to the identity of a bacterium. See Figure 7b below.

Procaryotes are known to exhibit a variety of types of tactic behavior, i.e., the ability to move (swim) in response to environmental stimuli. For example, during chemotaxis a bacterium can sense the quality and quantity of certain chemicals in its environment and swim towards them (if they are useful nutrients) or away from them (if they are harmful substances). Other types of tactic response in procaryotes include phototaxis, aerotaxis and magnetotaxis. The occurrence of tactic behavior provides evidence for the ecological (survival) advantage of flagella in bacteria and other procaryotes.

Detecting Bacterial Motility

Since motility is a primary criterion for the diagnosis and identification of bacteria, several techniques have been developed to demonstrate bacterial motility, directly or indirectly.

1. Flagellar stains outline flagella and show their pattern of distribution. If a bacterium possesses flagella, it is presumed to be motile.

Figure 7b. Flagellar stains of three bacteria a. Bacillus cereus b. Vibrio cholerae c. Bacillus brevis. CDC. Since the bacterial flagellum is below the resolving power of the light microscope, although bacteria can be seen swimming in a microscope field, the organelles of movement cannot be detected. Staining techniques such as Leifson's method utilize dyes and other components that precipitate along the protein filament and hence increase its effective diameter. Flagellar distribution is sometimes used to differentiate between morphologically related bacteria. For example, among the Gram-negative motile rod-shaped bacteria, the enterics have peritrichous flagella while the pseudomonads have polar flagella.

2. Motility test medium demonstrates if cells can swim in a semisolid medium. A semisolid medium such as O.75% agar is inoculated with the bacteria in a straight-line stab with a needle. After incubation, if turbidity (cloudiness) due to bacterial growth can be observed away from the line of the stab, it is evidence that the bacteria were able to swim through the medium.

Figure 8. Bacterial cultures grown in motility test medium. The tube on left is a non motile organism; the tube on right is a motile organism. Motility test medium is a semi-soft medium that is inoculated with a straight needle. If the bacteria  are motile, they will swim away from the line of inoculation in order to find nutrients, causing turbidity or cloudiness throughout the medium. If they are non motile, they will only grow along the line of inoculation. www.jlindquist.net/ generalmicro/dfmotility.html.


3. Direct microscopic observation of living bacteria in a wet mount. One must look for transient movement of swimming bacteria. Most unicellular bacteria, because of their small size, will shake back and forth in a wet mount observed at 400X or 1000X. This is Brownian movement, due to random collisions between water molecules and bacterial cells. True motility is confirmed by observing a bacterium swim from one side of the microscope field to the other side.



Figure 9.  Wet mount of the bacterium Rhodospirillum rubrum, about 1500X mag. Click here or on the image for a short video from the Department of Microbiology and Immunology, University of Leicester, that illustrates swimming motility of this photosynthetic purple bacterium.

Figure 10. A Desulfovibrio species. TEM. About 15,000X. The bacterium is motile by means of a single polar flagellum. Of course, one can detect flagella by means of electron microscopy. Perhaps this is an alternative way to determine bacterial motility, if you happen to have an electron microscope.

Fimbriae and Pili

Figure 11. Fimbriae (common pili) and flagella on the surface of bacterial cells. Left: dividing Shigella enclosed in fimbriae. The structures are probably involved in the bacterium's ability to adhere to the intestinal surface. Right: dividing pair of Salmonella displaying both its peritrichous flagella and its fimbriae. The fimbriae are much shorter and slightly smaller in diameter than flagella. Both Shigella and Salmonella are enteric bacteria that cause different types of intestinal diarrheas. The bacteria can be differentiated by a motility test. Salmonella is motile; Shigella is nonmotile.

Table 4. Some properties of pili and fimbriae.

The Cell Envelope

The cell envelope is a descriptive term for the several layers of material that envelope or enclose the protoplasm of the cell. The cell protoplasm (cytoplasm) is surrounded by the plasma membrane, a cell wall and a capsule. The cell wall itself is a layered structure in Gram-negative bacteria. All cells have a plasma membrane, which is the essential and definitive characteristic of a "cell". Almost all procaryotes have a cell wall to prevent damage to the underlying protoplast. Outside the cell wall, foremost as a surface structure, may be a polysaccharide capsule or  glycocalyx.

Figure 12. Profiles of the cell envelope the Gram-positive and Gram-negative bacteria. The Gram-positive wall is a uniformly thick layer external to the plasma membrane. It is composed mainly of peptidoglycan (murein). The Gram-negative wall appears thin and multilayered. It consists of a relatively thin peptidoglycan sheet between the plasma membrane and a phospholipid-lipopolysaccharide outer membrane. The space between the inner (plasma) and outer membranes (wherein the peptidoglycan resides) is called the periplasm.

Capsules

Most bacteria contain some sort of a polysaccharide layer outside of the cell wall polymer. In a general sense, this layer is called a capsule. A true capsule is a discrete detectable layer of polysaccharides deposited outside the cell wall. A less discrete structure or matrix which embeds the cells is a called a slime layer or a biofilm. A type of capsule found in bacteria called a glycocalyx or microcapsule is a very thin layer of tangled polysaccharide fibers on the cell surface.

Figure 13. Bacterial capsules outlined by India ink viewed by light microscopy. This is a true capsule, a discrete layer of polysaccharide surrounding the cells. Sometimes bacterial cells are embedded more randomly in a polysaccharide matrix called a slime layer or biofilm. Polysaccharide films that may inevitably be present on the surfaces of bacterial cells, but which cannot be detected visually, are called glycocalyx.
 

Figure 14. Negative stain of Streptococcus pyogenes viewed by transmission electron microscopy (28,000X). The halo around the chain of cells is the hyaluronic acid capsule that surrounds the exterior of the bacteria. The septa between dividing pairs of cells may also be seen. Electron micrograph of Streptococcus pyogenes by Maria Fazio and Vincent A. Fischetti, Ph.D. with permission. The Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University.

Capsules are generally composed of polysaccharides; rarely they contain amino sugars or peptides.

Capsules have several functions and often have multiple functions in a particular organism. Like fimbriae, capsules, slime layers, and glycocalyx often mediate adherence of cells to surfaces. Capsules also protect bacterial cells from engulfment by predatory protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin. Capsules in certain soil bacteria protect cells from perennial effects of drying or desiccation. Capsular materials (e.g. dextrans) may be overproduced when bacteria are fed sugars to become reserves of carbohydrate for subsequent metabolism.

Figure 15. Colonies of Bacillus anthracis. CDC. The slimy or mucoid appearance of a bacterial colony is usually evidence of capsule production. In the case of B. anthracis, the capsule is composed of poly-D-glutamate. The capsule is an essential determinant of virulence to the bacterium. In the early stages of colonization and infection the capsule protects the bacteria from assaults by the immune and phagocytic systems.

Figure 16 (Left) Dental plaque revealed by a harmless red dye. http://www.medicdirect.co.uk/DentalHealth (Right)  Human dental plaque. Transmission electron micrograph by Marilee Sellers, Northern Arizona University. http://www4.nau.edu/electron/TEM_img.htm

Another important characteristic of capsules may be their ability to block some step in the phagocytic process and thereby prevent the bacterial cells from being engulfed or destroyed by phagocytes (white blood cells). For example, the human pathogens Streptococcus pneumoniae (lobar pneumonia), Bacillus anthracis (anthrax), and  Neisseria meningitidis (meningitis) are each able to resist attack by phagocytes because they cover their surfaces with some type of capsular material.

Table 5.  Functions of Capsules, Glycocalyx, Slime Layers and Biofilms

Cell Wall

The cell walls of bacteria deserve special attention for several reasons:
1. They are an essential structure for viability, as described above.
2. They are composed of unique components found nowhere else in nature.
3. They are one of the most important sites for attack by antibiotics.
4. They provide ligands for adherence and receptor sites for drugs or viruses.
5. They cause symptoms of disease in animals.
6. They provide for immunological distinction and immunological variation among strains of bacteria.

Most procaryotes have a rigid cell wall. The cell wall is an essential structure that protects the cell protoplast (the region bound by and including the membrane) from mechanical damage and from osmotic rupture or lysis. Bacteria usually live in relatively dilute environments such that the accumulation of solutes inside the cell cytoplasm greatly exceeds the total solute concentration in the outside environment. Thus, the osmotic pressure against the inside of the plasma membrane may be the equivalent of 10-25 atmospheres. Since the membrane is a delicate, plastic structure, it must be restrained by an outside wall made of porous, rigid material that has high tensile strength. Such a material is murein, the ubiquitous component of bacterial cell walls.

Bacterial murein is a unique type of peptidoglycan. Peptidoglycan is a polymer of sugars (a glycan) cross-linked by short chains of amino acids (peptide). All bacterial peptidoglycans contain N-acetylmuramic acid, which is the definitive component of murein. The cell walls of archaea may be composed of protein, polysaccharides, or peptidoglycan-like molecules, but never do they contain murein. This feature distinguishes the bacteria from the archaea.

The profiles of the cell surface of bacteria, as seen with the electron microscope, are drawn in Figure 12. In  Gram-positive Bacteria (those that retain the purple crystal violet dye when subjected to the Gram-staining procedure) the cell wall is thick (15-80 nanometers), consisting of several layers of peptidoglycan. Running perpendicular to the peptidoglycan sheets are a group of molecules called teichoic acids which are unique to the Gram-positive cell wall.

Figure 17.  Structure of the Gram-positive bacterial cell wall. The wall is relatively thick and consists of many layers of peptidoglycan interspersed with teichoic acids that run perpendicular to the peptidoglycan sheets.

In the Gram-negative Bacteria (which do not retain the crystal violet in the Gram-stain procedure) the cell wall is relatively thin (10 nanometers) and is composed of a single layer of peptidoglycan surrounded by a membranous structure called the outer membrane. The outer membrane of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS or endotoxin), which is toxic to animals. In Gram-negative bacteria the outer membrane is usually considered as part of the cell wall.

Figure 18. Structure of the Gram-negative cell wall. The wall is relatively thin and contains much less peptidoglycan than the Gram-positive wall. Also, teichoic acids are absent. However, the Gram negative cell wall consists of an outer membrane that is outside of the peptidoglycan layer. The outer membrane is attached to the peptidoglycan sheet by a unique group of lipoprotein molecules.

In E. coli and other Gram-negative bacteria, the glycan backbone of peptidoglycan is made up of alternating molecules of N-acetylglucosamine (G) and N-acetylmuramic acid (M). The N-acetylmuramic acid (M) attaches a peptide side chain that contains the amino acids L-alanine, (L-ala), D-glutamate (D-glu), Diaminopimelic acid (DAP), and D-alanine (D-ala). The muramic acid subunit of E. coli is shown in Figure 19 below.

Figure 19. The structure of peptidoglycan of Escherichia coli. a. The glycan backbone is a repeat polymer of two amino sugars, N-acetylglucosamine (G) and N-acetylmuramic acid (M). Attached to the N-acetylmuramic acid is a tetrapeptide side chain consisting of L-ala-D-glu-DAP-D-ala.  b. Abbreviated structure of the muramic acid subunit.  c. Nearby tetrapeptide side chains may be linked to one another by an interpeptide bond between DAP on one chain and D-ala on the other.  d. The polymeric form of the molecule.

The glycan backbone of the peptidoglycan molecule can be cleaved by an enzyme called lysozyme that is present in animal serum, tissues and secretions, and in phagocyte granules. The function of lysozyme is to lyse (rupture) bacterial cells as a defense against bacterial pathogens. Some Gram-positive bacteria are very sensitive to lysozyme and the enzyme is quite active at low concentrations. Lachrymal secretions (tears) can be diluted 1:40,000 and retain the ability to lyse certain bacterial cells. Gram-negative bacteria are less vulnerable to attack by lysozyme because their peptidoglycan is shielded by the outer membrane. The exact site of lysozymal cleavage is the beta 1,4 bond between N-acetylmuramic acid (M) and N-acetylglucosamine (G).

The formation of the peptide bond between nearby chains of peptidoglycan is blocked by a group of antibiotics of the  beta lactam class, which includes penicillin and cephalosporin and their relatives. Hence, the beta lactam antibiotics are effective against many bacteria because they prevent the assembly of the bacterial cell wall. In the presence of these antibiotics, the bacterium grows and synthesizes cell wall material but is unable to assemble the peptidoglycan sheet. Hence, the wall becomes progressively weaker and weaker until the cell lyses or ruptures. The cell wall is one of the most effective targets in bacterial cells for antibiotics because the wall material is unique to bacteria, and the animal taking the antibiotic totally lacks the target against which the antibiotic is directed.




Figure 20. Assembly of the Gram-negative peptidoglycan. The site of action of lysozyme is to break the bond that links G to M. The site of action of action of penicillin is to prevent the formation of the interpeptide bond that that occasionally joins the peptide chains to one another. In either case, the result is the lysis of the bacterial cell.

In Gram-positive bacteria there are numerous different peptide arrangements among peptidoglycans. The best studied is the murein of Staphylococcus aureus shown in Figure 21 below. The most significant difference in the Gram-positive wall is the occurrence of an interpeptide bridge of amino acids that connects nearby side chains to one another. Assembly of the interpeptide bridge in Gram-positive murein is inhibited by the beta lactam antibiotics in the same manner as the interpeptide bond in Gram-negative murein. Gram-positive bacteria are more sensitive to penicillin than Gram-negative bacteria because the peptidoglycan is not protected by an outer membrane and it is a more abundant molecule.

Figure 21. Schematic diagram of the peptidoglycan sheet of Staphylococcus aureus. G = N-acetyl-glucosamine; M = N-acetyl-muramic acid; L-ala = L-alanine; D-ala = D-alanine; D-glu = D-glutamine; L-lys = L-lysine. This is one type of murein found in Gram-positive bacteria.

Figure 22. Assembly of Gram-positive peptidoglycan. The main difference between Gram-negative and Gram-positive peptidoglycans is the occurrence of a bridge of amino acids in the latter which links the peptide side chains to one another. In Gram-positive bacteria, this is referred to as the interpeptide bridge. The site of action of lysozyme and penicillin are similar in Gram-positive and Gram-negative bacteria, except in Gram-positives it is the assembly of the interpeptide bridge (rather than the interpeptide bond) that is blocked by the beta lactam antibiotics.


The Outer Membrane of Gram-negative Bacteria

Of special interest as a component of the Gram-negative cell wall is the outer membrane, a discrete bilayered structure on the outside of the peptidoglycan sheet (see Figure 12 above and Figure 19 below). For the bacterium, the outer membrane is first and foremost a permeability barrier, but primarily due to its lipopolysaccharide content, it possesses many interesting and important characteristics of Gram-negative bacteria. The outer membrane superficially resembles the plasma membrane except the outer face contains a unique type of Lipopolysaccharide referred to by medical microbiologists as endotoxin because of its toxic effects in animals.

Figure 23. Schematic illustration of the outer membrane, cell wall,  and plasma membrane of a Gram-negative bacterium. Lipopolysaccharide (LPS or endotoxin) is located on the outer face of the outer membrane.

Figure 24. Structure of bacterial lipopolysaccharide or endotoxin. The lipo- part (Lipid A) is the toxic portion of the molecule. It causes fever, inflammation, hemorrhage and shock in animals. The -polysaccharide part of the molecule is responsible for antigenic properties of the bacterium, which influences how the animal immune system will respond.

Bacterial lipopolysaccharides are toxic to animals. When injected in small amounts LPS or endotoxin activates several host responses that lead to fever, inflammation and shock. Endotoxins may play a role in infection by any Gram-negative bacterium. The toxic component of endotoxin (LPS) is Lipid A. The O-specific polysaccharide may provide for adherence or resistance to phagocytosis, in the same manner as fimbriae and capsules. The O polysaccharide (also referred to as the O antigen) also accounts for multiple antigenic types (serotypes) among Gram-negative bacterial pathogens. Thus, E. coli O157 (the Jack-in-the-Box and Stock Pavilion E. coli) is #157 of the different antigenic types of E. coli and may be identified on this basis.

The Gram stain and bacterial cell walls

A correlation between Gram stain reaction and cell wall properties of bacteria is summarized in Table 5. The Gram stain procedure contains a "destaining" step wherein the cells are washed with an acetone-alcohol mixture. The lipid content of the Gram-negative wall probably affects the outcome of this step so that Gram-positive cells retain a primary stain while Gram-negative cells are destained.

Table 5. Correlation of the Grams stain with cell wall properties of Bacteria.

(1) A few Gram-negative bacteria are sensitive to natural penicillins. Many Gram-negative bacteria are sensitive to some type of penicillin, especially semisynthetic penicillins. Gram-negative bacteria, including E. coli, can be made sensitive to natural penicillin by procedures that disrupt the permeability characteristics of the outer membrane.
(2) Gram-negative bacteria are sensitive to lysozyme if pretreated by some procedure that removes the outer membrane and exposes the peptidoglycan directly to the enzyme.

Cell Wall-less Forms

A few bacteria are able to live or exist without a cell wall. The mycoplasmas are a group of bacteria that lack a cell wall. Mycoplasmas have sterol-like molecules incorporated into their membranes and they are usually inhabitants of osmotically-protected environments (contain a high concentration of external solute). Mycoplasma pneumoniae is the cause of primary atypical bacterial pneumonia, known in the vernacular as "walking pneumonia". For obvious reasons, penicillin is ineffective in treatment of this type of pneumonia. Sometimes, under the pressure of antibiotic therapy, pathogenic streptococci can revert to cell wall-less forms (called spheroplasts) and persist or survive in osmotically-protected tissues. When the antibiotic is withdrawn from therapy the organisms may regrow their cell walls and reinfect unprotected tissues.

The Cytoplasmic Membrane

 The cytoplasmic membrane of bacterial cells is a delicate and plastic structure that completely encloses the cell cytoplasm (or protoplasm). The bacterial membrane is composed of 40 percent phospholipid and 60 percent protein. The phospholipids are amphoteric molecules, meaning they have a water-soluble hydrophilic region (the glycerol "head") attached to two insoluble hydrophobic fatty acid "tails". In water, such molecules naturally form the molecular bilayer characteristic of membranes. The fatty acid tails from one layer face towards the fatty acid tails of the second layer ("likes dissolve like"), and the glycerol heads naturally turn towards the water (Figure 25 and 26).  Dispersed throughout the bilayer are various structural and enzymatic proteins which carry out most membrane functions. This arrangement of proteins and phospholipids forms what is called the fluid mosaic membrane as illustrated in Figure 27.

Figure 25. Molecular structure of a phospholipid, the building block of membranes. Inset - the usual depiction of a membrane phospholipid containing a phosphatidyl-glycerol "head" attached to two fatty acid "tails".

Figure 26. Organization of phospholipids in aqueous solution to form a bilayer. The hydrophilic phosphatidyl glycerols form the inner and outer faces of the membrane. The fatty acids orient themselves towards one another to form the hydrophobic interior of the membrane.

Figure 27. Fluid mosaic model of a biological membrane. In aqueous environments membrane phospholipids arrange themselves in such a way that they spontaneously form a fluid bilayer. Membrane protein may be either structural or functional. Proteins may be permanently or transiently associated with one side or the other of the membrane or built into the bilayer, or they may span the bilayer forming transport channels through the membrane.  

Functions of the Cytoplasmic Membrane

Permeability Barrier
The cell membrane is the most dynamic structure in the cell. Its main function is as a permeability barrier that regulates the passage of substances into and out of the cell. The plasma membrane is the definitive structure of a cell since it sequesters the molecules of life in the cytoplasm, separating it from the outside environment. The bacterial membrane freely allows passage of water and a few small uncharged molecules (less than molecular weight of 100 daltons), but it does not allow passage of larger molecules or any charged substances except when monitored by proteins in the membrane called transport systems.

Transport of Solutes
The presence of transport systems in the membranes allows the bacteria to accumulate solutes and chemical precursors of cell material inside their cytoplasm at concentrations which greatly exceed the concentrations in the environment. Remember, most bacteria live in relatively dilute environments (e.g. a lake or stream) where the concentration of the business molecules of life is greater inside of the cell than in the environment. Hence, the bacterial cells must transport their nutrients from the environment and maintain a higher concentration of solutes inside the cell than outside the cell. This comes at a price. To concentrate a substance against the environmental gradient using a membrane transport system always costs energy in one form or another.

Bacteria have a variety of types of transport systems which can be used alternatively in various environmental situations. The most important transport systems are called active transport systems since they require energy and concentrate substances inside of the cell. At least 80 percent of the molecules needed in the cytoplasm are taken up by the process of active transport. Active transport systems are mediated by proteins in the membrane called carrier proteins or "permeases" that are generally quite specific for the substances that they will transport. All active transport systems require energy to operate. Some use chemical energy derived from ATP; others use proton motive force (pmf), which is derived from the establishment of a charge and a pH gradient on opposite sides of the membrane.

Figure 28. Operation of bacterial transport systems. Bacterial transport systems are operated by membrane proteins, also called carriers or permeases. Facilitated diffusion is a carrier-mediated system that does not require energy and does not concentrate solutes against a gradient. Active transport systems use energy and are able to concentrate molecules against a concentration gradient. Group translocation systems also use chemical energy during transport but they are distinguished from active transport because they modify the solute during its passage across the membrane. Most solutes in bacteria are transported by active transport systems.

Besides transport proteins that selectively mediate the passage of substances into and out of the cell, bacterial membranes may also contain sensing proteins that measure concentrations of molecules in the environment or binding proteins that translocate signals from the environment to genetic and metabolic machinery in the cytoplasm.

Generation of Energy

Unlike eucaryotes, bacteria don't have intracellular organelles for energy producing processes such as respiration or photosynthesis. Instead, the cytoplasmic membrane carries out these functions. The membrane is the location of electron transport systems (ETS) used to produce energy during photosynthesis and respiration, and it is the location of an enzyme called ATP synthetase  (ATPase) which is used to synthesize ATP.

When the electron transport system operates, it establishes a pH gradient across of the membrane due to an accumulation of protons (H⁺) outside and hydroxyl ion (OH^-) inside. Thus the outside is acidic and the inside is alkaline. Operation of the ETS also establishes a charge on the membrane called proton motive force  (pmf). The outer face of the membrane becomes charged positive while inner face is charged negative, so the membrane has a positive side and a negative side, like a battery. The pmf can be used to do various types of work including the rotation of the flagellum, or active transport as described above. The pmf can also be used to make ATP by the membrane ATPase enzyme which consumes protons when it synthesizes ATP from ADP and phosphate. The connection between electron transport, establishment of pmf, and ATP synthesis during respiration is known as oxidative phosphorylation; during photosynthesis, it is called phosphorylation.

Figure 29 below illustrates the membrane of E. coli. The topographical features of the membrane from top to bottom are 1. lactose transport system; 2. the flagellar motor coupled to the hook and filament; 3. Na⁺ transport (export) system;  4. Ca⁺⁺  transport (export) system; 5. electron transport system; 6. ATPase enzyme; 7. proline transport system. The operation ot the electron transport system during respiration produces the H⁺ charge on the membrane (pmf). The pmf (H⁺) is used by the transport systems to move molecules from one side of the membrane to the other; by the flagellar motor ring to rotate the flagellar filament; and by the ATPase enzyme to synthesize ATP.


Figure 29. Schematic view of the cytoplasmic membrane of Escherichia coli. Various transport systems are shown which operate as active transport systems. The rings which constitute the flagellar motor are shown. The motor ring is imbedded in the phospholipid bilayer. It is powered by pmf to to rotate the flagellar filament. The electron transport system is shown oxidizing NAD by removal of a pair of electrons, passing them through its sequence of carriers eventually to O₂. ATPase is the transmembranous protein enzyme that utilizes protons from the outside to synthesize ATP on the inside of the membrane.

In bacteria, the photosynthetic pigments that harvest light energy for conversion into chemical energy are also located in the membrane.  Membranes may contain other enzymes involved in many metabolic processes such as cell wall synthesis, septum formation, membrane synthesis, DNA replication, CO₂ fixation and ammonia oxidation. The predominant functions of bacterial membranes are listed in the table below.

Table 6. Functions of the procaryotic plasma membrane.

1. Osmotic or permeability barrier.

2. Location of transport systems for specific solutes (nutrients and ions).

3. Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and ATP-synthesizing ATPase
 
4. Synthesis of membrane lipids (including lipopolysaccharide in Gram-negative cells)

5. Synthesis of murein (cell wall peptidoglycan)

6. Coordination of DNA replication and segregation with septum formation and cell division

7. Location of specialized enzyme systems, such as for CO₂ fixation, nitrogen fixation

The Cytoplasm

The cytoplasm of bacterial cells consists consists of an aqueous solution of three groups of molecules: macromolecules such as proteins (enzymes), DNA, mRNA and tRNA;  small molecules that are energy sources, precursors of macromolecules, metabolites or vitamins (see Table 6); various inorganic ions and cofactors (see Table 7). The cytoplasm of procaryotes is more gel-like than that of eucaryotes and the processes of cytoplasmic streaming, which are evident in eucaryotes, do not occur.

Table 6. Small molecules present in a growing bacterial cell.

Table 7. Inorganic ions present in a growing bacterial cell.

The cytoplasmic constituents of bacterial cells invariably include the procaryotic chromosome (nucleoid), ribosomes, and several hundred proteins and enzymes. The chromosome is typically one large circular molecule of DNA, more or less free in the cytoplasm. Procaryotes sometimes possess smaller extrachromosomal pieces of DNA called plasmids. The total DNA content of a procaryote is referred to as the cell genome. The cell chromosome is the genetic control center of the cell which determines all the properties and functions of the bacterium. During cell growth and division, the procaryotic chromosome is replicated in to make an exact copy of the molecule for distribution to progeny cells. 

Figure 30. When a bacterium such as E. coli is "gently lysed" the chromosomal DNA  leaks out of the cell as a continuous molecule that is many times longer than the length of the cell.

The distinct granular appearance of procaryotic cytoplasm is due to the presence and distribution of ribosomes. The ribosomes of procaryotes are smaller than cytoplasmic ribosomes of eucaryotes. Procaryotic ribosomes are 70S in size, being composed of 30S and 50S subunits. The 80S ribosomes of eucaryotes are made up of 40S and 60S subunits. Ribosomes are involved in the process of translation (protein synthesis), but some details of their activities differ in eucaryotes, bacteria and archaea. Protein synthesis using 70S ribosomes occurs in eucaryotic mitochondria and chloroplasts, and this is taken as a major line of evidence that these organelles are descended from procaryotes.


Figure 31. The bacterial chromosome or nucleoid is the nonstaining region in the interior of the cell cytoplasm. The granular structures distributed throughout the cytoplasm are cell ribosomes.

Inclusions

Often contained in the cytoplasm of procaryotic cells is one or another of some type of inclusion granule. Inclusions are distinct granules that may occupy a substantial part of the cytoplasm. Inclusion granules are usually reserve materials of some sort. For example, carbon and energy reserves may be stored as glycogen (a polymer of glucose) or as polybetahydroxybutyric acid (a type of fat) granules. Polyphosphate inclusions are reserves of PO₄ and possibly energy; elemental sulfur (sulfur globules) are stored by some phototrophic and some lithotrophic procaryotes as reserves of energy or electrons. Some inclusion bodies are actually membranous vesicles or intrusions into the cytoplasm which contain photosynthetic pigments or enzymes.

Table 8. Some inclusions in bacterial cells.

Figure 32. A variety of bacterial inclusions.  a. PHB granules; b. a parasporal BT crystal in the sporangium of Bacillus thuringiensis; c. carboxysomes in Anabaena viriabilis, showing their polyhedral shape; d. sulfur globules in the cytoplasm of Beggiatoa. 

A bacterial structure sometimes observed as an inclusion is actually a type of dormant cell called an endospore. Endospores are formed by a few groups of Bacteria as intracellular structures, but ultimately they are released as free endospores. Biologically, endospores are a fascinating type of cell. Endospores exhibit no signs of life, being described as cryptobiotic. They are highly resistant to environmental stresses such as high temperature (some endospores can be boiled for hours and retain their viability), irradiation, strong acids, disinfectants, etc. They are probably the most durable cell produced in nature. Although cryptobiotic, they retain viability indefinitely such that under appropriate environmental conditions, they germinate back into vegetative cells. Endospores are formed by vegetative cells in response to environmental signals that indicate a limiting factor for vegetative growth, such as exhaustion of an essential nutrient. They germinate and become vegetative cells when the environmental stress is relieved. Hence, endospore-formation is a mechanism of survival rather than a mechanism of reproduction.

Figure 33. Early and late stages of endospore formation. Drawing by Vaike Haas, University of Wisconsin Madison. During endospore formation, a vegetative cell is converted to a heat-resistant spore. There are eight stages, O,I-VII, in the sporulation cycle of a Bacillus species, and the process takes about eight hours. During the early stages (Stage II,) one bacterial chromosome and a few ribosomes are partitioned off by the bacterial membrane to form a protoplast within the mother cell. By the late stages (Stage VI) the protoplast (now called a forespore) has developed a second membrane and several wall-like layers of material are deposited between the two membranes.

Table 9. Differences between endospores and vegetative cells.

Figure 34. Bacterial endospores. Phase microscopy of sporulating bacteria demonstrates the refractility of endospores, as well as characteristic spore shapes and locations within the mother cell.

Figure 35. Electron micrograph of a bacterial endospore. The spore has a core wall of unique peptidoglycan surrounded by several layers, including the cortex, the spore coat and the exosporium. The dehydrated core contains the bacterial chromosome and a few ribosomes and enzymes to jump-start protein synthesis and metabolism during germination.