Todar's Online Textbook of Bacteriology 

STRUCTURE AND FUNCTION OF PROCARYOTIC CELLS

© 2008 Kenneth Todar University of Wisconsin-Madison Department of Bacteriology
 

Drawing of a typical bacterial cell, by Vaike Haas, University of Wisconsin-Madison

Primary Structure of Biological Macromolecules Determines Function

Procaryotic structural components consist of macromolecules such as DNA, RNA, proteins, polysaccharides, phospholipids, or some combination thereof. The macromolecules are made up of primary subunits such as nucleotides, amino acids and sugars (Table 1). It is the sequence in which the subunits are put together in the macromolecule, called the primary structure, that determines many of the properties that the macromolecule will have. Thus, the genetic code is determined by specific nuleotide base sequences in chromosomal DNA; the amino acid sequence in a protein determines the properties and function of the protein; and sequence of sugars in bacterial lipopolysaccharides determines unique cell wall properties for pathogens.  The primary structure of a macromolecule will drive its function, and differences within the primary structure of biological macromolecules accounts for the immense diversity of life.

 Table 1. Macromolecules that make up cell material

Macromolecule

Primary Subunits

Where found in cell

Proteins

amino acids

Flagella, pili, cell walls, cytoplasmic membranes, ribosomes, cytoplasm

Polysaccharides

sugars (carbohydrates)

capsules, inclusions (storage), cell walls

Phospholipids

fatty acids

membranes

Nucleic Acids
(DNA/RNA)

 nucleotides

DNA: nucleoid (chromosome), plasmids
rRNA: ribosomes; mRNA, tRNA: cytoplasm

Procaryotic Cell Architecture

At one time it was thought that bacteria and other procaryotes 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. Procaryotes are cells of relatively simple construction, especially if compared to eucaryotes. Whereas eucaryotic cells have a preponderance of organelles with separate cellular functions, procaryotes carry out all cellular functions as individual units.

A procaryotic cell has five essential structural components: a nucleoid (DNA), ribosomes, cell membrane, cell wall, and some sort of surface layer, which may or may not be an inherent part of the wall.

Structurally, there are 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 (Figure 1). 


Figure 1. Cutaway drawing of a typical bacterial cell illustrating structural components.  See Table 2 below for chemical composition and function of the labeled components.

Table 2. Summary of characteristics of typical bacterial cell structures
Structure
Flagella 		Function(s)

Swimming movement

 Predominant chemical composition

Protein
Pili
Sex pilus Stabilizes mating bacteria during DNA transfer by conjugation Protein
Common pili or fimbriae Attachment to surfaces; protection against phagotrophic engulfment Protein 
Capsules (includes "slime layers" and glycocalyx) Attachment to surfaces; protection against phagocytic engulfment, occasionally killing or digestion; reserve of nutrients or protection against desiccation Usually polysaccharide; occasionally polypeptide
Cell wall
Gram-positive bacteria Prevents osmotic lysis of cell protoplast and confers rigidity and shape on cells Peptidoglycan (murein) complexed with teichoic acids
Gram-negative bacteria Peptidoglycan prevents osmotic lysis and confers rigidity and shape; outer membrane is permeability barrier; associated LPS and proteins have various functions Peptidoglycan (murein) surrounded by phospholipid protein-lipopolysaccharide "outer membrane"
Plasma membrane Permeability barrier; transport of solutes; energy generation; location of numerous enzyme systems Phospholipid and protein
Ribosomes Sites of translation (protein synthesis) RNA and protein
Inclusions Often reserves of nutrients; additional specialized functions Highly variable; carbohydrate, lipid, protein or inorganic
Chromosome Genetic material of cell  DNA
Plasmid Extrachromosomal genetic material DNA



Figure 2 . Electron micrograph of an ultra-thin section of a dividing pair of group A streptococci (20,000X). The cell surface fimbriae (fibrils) are evident. The bacterial cell wall is 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.


Appendages: flagella, fimbriae and pili

 
Salmonella enterica. TEM about 10,000X. 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, and they lack the typical "9 + 2" arrangement of microtubules. 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. Bacterial flagella are powered by proton motive force (chemiosmotic potential) established on the bacterial membrane, rather than ATP hydrolysis which powers eucaryotic flagella. About half of the bacilli and all of the spiral and curved bacteria are motile by means of flagella. Very few cocci are motile, which reflects their adaptation to dry environments and their lack of hydrodynamic design.

The ultrastructure of the flagellum of E. coli is illustrated in Figure 3 below (after Dr. Julius Adler of the University of Wisconsin). About 50 genes are required for flagellar synthesis and function. The flagellar apparatus consists of several distinct proteins: a system of rings embedded 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. The outermost rings, the P and L rings, located in the periplasm and the outer membrane respectively, function as bushings to support the rod where it is joined to the hook of the filament on the cell surface. As the M ring turns, powered by an influx of protons, the rotary motion is transferred to the filament which turns to propel the bacterium.


Figure 3. 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 outer rings in the peptidoglycan and outer membrane are support rings or "bushings" for the rod. The filament rotates and contracts which propels and steers the cell during movement. Compare with Figure  below.

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, Pseudomonas has polar flagella to distinguish them from enteric bacteria, which have peritrichous flagella.


Figure 4. 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 6 below.

Flagella were proven to be organelles of bacterial motility by shearing them off (by mixing cells in a blender) and observing that the cells could no longer swim although they remained viable. As the flagella were re-grown and reached a critical length, swimming movement was restored to the cells. The flagellar filament grows at its tip (by the deposition of new protein subunits) not at its base (like a hair).

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 5. 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 occasionally 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 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.
 
OFF THE WALL. Julius Adler exploited this observation during his studies of chemotaxis in E. coli. He prepared a gradient of glucose by allowing the sugar to diffuse into a semisolid medium from a central point in the medium. This established a concentration gradient of glucose along the radius of diffusion. When E. coli cells were seeded in the medium at the lowest concentration of glucose (along the edge of the circle), they swam up the gradient towards a higher concentration (the center of the circle), exhibiting their chemotactic response to swim towards a useful nutrient. Later, Adler developed a tracking microscope that could record and film the track that E. coli takes as it swims towards a chemotactic attractant or away from a chemotactic repellent. This led to an understanding of the mechanisms of bacterial chemotaxis, first at a structural level, then at a biomolecular level.

Figure 6. 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 the bacterium swim from one side of the microscope field to the other side.


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 7. A Desulfovibrio species. TEM. About 15,000X. The bacterium is motile by means of a single polar flagellum. Of course, one can determine the presence of 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

Fimbriae and pili are interchangeable terms used to designate short, hair-like structures on the surfaces of procaryotic cells. Like flagella, they are composed of protein. Fimbriae are shorter and stiffer than flagella, and slightly smaller in diameter. Generally, fimbriae have nothing to do with bacterial movement (there are exceptions, e.g. twitching movement on Pseudomonas). Fimbriae are very common in Gram-negative bacteria, but occur in some archaea and Gram-positive bacteria as well. Fimbriae are most often involved in adherence of bacteria to surfaces, substrates and other cells or tissues in nature. In E. coli, a specialized type of pilus, the F or sex pilus, apparently stabilizes mating bacteria during the process of conjugation, but the function of the smaller, more numerous common pili is quite different.

Common pili (almost always called fimbriae) are usually involved in specific adherence (attachment) of procaryotes to surfaces in nature. In medical situations, they are major determinants of bacterial virulence because they allow pathogens to attach to (colonize) tissues and/or to resist attack by phagocytic white blood cells. For example, pathogenic Neisseria gonorrhoeae adheres specifically to the human cervical or urethral epithelium by means of its fimbriae; enterotoxigenic strains of E. coli adhere to the mucosal epithelium of the intestine by means of specific fimbriae; the M-protein and associated fimbriae of Streptococcus pyogenes (See Figure 2) are involved in adherence and to resistance to engulfment by phagocytes.


Figure 8. 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 3. Some properties of pili and fimbriae
Bacterial species where observed Typical number on cell Distribution on cell surface Function
Escherichia coli (F or sex pilus) 1-4 uniform stabilizes bacteria during transfer of DNA during conjugation
Escherichia coli (common pili or Type 1 fimbriae) 100-200 uniform surface adherence to epithelial cells of the GI tract
Neisseria gonorrhoeae 100-200 uniform surface adherence to epithelial cells of the urogenital tract
Streptococcus pyogenes (fimbriae plus the M-protein) ? uniform adherence, resistance to phagocytosis; antigenic variability
Pseudomonas aeruginosa 10-20 polar surface adherence
Sulfolobus acidocaldarius
(an archaic)		 ? ? attachment to sulfur particles


The Cell Envelope: capsules, cell walls and cell membranes

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 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 9. 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 procaryotes 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 is a thin layer of tangled polysaccharide fibers which occurs on  surface of cells growing in nature (as opposed to the laboratory). Some microbiologists refer to all capsules as glycocalyx and do not differentiate microcapsules.


Figure 10. 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 11. 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 polysaccharide; rarely they contain amino sugars or peptides (Table 4).

Table 4. Chemical composition of some bacterial capsules
Bacterium Capsule composition Structural subunits
Gram-positive Bacteria

Bacillus anthracis polypeptide (polyglutamic acid) D-glutamic acid
Bacillus megaterium polypeptide and polysaccharide D-glutamic acid, amino sugars, sugars
Streptococcus mutans polysaccharide (dextran) glucose
Streptococcus pneumoniae polysaccharides sugars, amino sugars, uronic acids
Streptococcus pyogenes polysaccharide (hyaluronic acid) N-acetyl-glucosamine and glucuronic acid
Gram-negative Bacteria

Acetobacter xylinum polysaccharide (cellulose) glucose
Escherichia coli polysaccharide (colonic acid) glucose, galactose, fucose glucuronic acid
Pseudomonas aeruginosa polysaccharide mannuronic acid
Azotobacter vinelandii polysaccharide glucuronic acid
Agrobacterium tumefaciens polysaccharide (glucan) glucose

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 12. Colonies of Bacillus anthracis. 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.

Some bacteria produce slime materials to adhere and float themselves as colonial masses in their environments. Other bacteria produce slime materials to attach themselves to a surface or substrate. Bacteria may attach to surface, produce slime, divide and produce microcolonies within the slime layer, and construct a biofilm, which becomes an enriched and protected environment for themselves and other bacteria.

A classic example of biofilm construction in nature is the formation of dental plaque mediated by the oral bacterium, Streptococcus mutans. The bacteria adhere specifically to the pellicle of the tooth by means of a protein on the cell surface. The bacteria grow and synthesize a dextran capsule which binds them to the enamel and forms a biofilm some 300-500 cells in thickness. The bacteria are able to cleave sucrose (provided by the animal diet) into glucose plus fructose. The fructose is fermented as an energy source for bacterial growth. The glucose is polymerized into an extracellular dextran polymer that cements the bacteria to tooth enamel and becomes the matrix of dental plaque. The dextran slime can be depolymerized to glucose for use as a carbon source, resulting in production of lactic acid within the biofilm (plaque) that decalcifies the enamel and leads to dental caries or bacterial infection of the tooth.

Figure 13. (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 bacterial cells from being engulfed or destroyed by phagocytes. For example, the primary determinant of virulence of the pathogen Streptococcus pneumoniae is its polysaccharide capsule, which prevents ingestion of pneumococci by alveolar macrophages. Bacillus anthracis survives phagocytic engulfment because the lysosomal enzymes of the phagocyte cannot initiate an attack on the poly-D-glutamate capsule of the bacterium. Bacteria such as Pseudomonas aeruginosa, that construct a biofilm made of extracellular slime when colonizing tissues, are also resistant to phagocytes, which cannot penetrate the biofilm.

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 from mechanical damage and from osmotic rupture or lysis. Procaryotes usually live in relatively dilute environments such that the accumulation of solutes inside the procaryotic 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 atm. 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.

Murein is a unique type of peptidoglycan, a polymer of disaccharides (glycan) cross-linked by short chains of amino acids (peptide). Many types of peptidoglycan exist. 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.

In the Gram-positive Bacteria (those that retain the purple crystal violet dye when subjected to the Gram-staining procedure), the cell wall consists of several layers of peptidoglycan. Running perpendicular to the peptidoglycan sheets is a group of molecules called teichoic acids which are unique to the Gram-positive cell wall (Figure 14).
 
Figure 14.  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), the cell wall 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 thought of as part of the cell wall (Figure 15).


Figure 15. 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 the Gram-positive Bacteria, the cell wall is thick (15-80 nanometers), consisting of several layers of peptidoglycan. In the Gram-negative Bacteria the cell wall is relatively thin (10 nanometers) and is composed of a single layer of peptidoglycan surrounded by an outer membrane.

Peptidoglycan structure and arrangement in E. coli is representative of all Enterobacteriaceae, as well as many other Gram-negative bacteria. The glycan backbone is made up of alternating molecules of N-acetylglucosamine (G) and N-acetylmuramic acid (M) connected by a beta 1,4-glycoside bond. The 3-carbon of N-acetylmuramic acid (M) is substituted with a lactyl ether group derived from pyruvate. The lactyl ether connects the glycan backbone to a peptide side chain that contains L-alanine, (L-ala), D-glutamate (D-glu), Diaminopimelic acid (DAP), and D-alanine (D-ala). MurNAc is unique to bacterial cell walls, as is D-glu, DAP and D-ala. The muramic acid subunit of E. coli is shown in Figure 16 below.


Figure 16. The structure of the muramic acid subunit of the peptidoglycan of Escherichia coli. This is the type of murein found in most Gram-negative bacteria. 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 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.

Strands of murein are assembled in the periplasm from about 10 muramic acid subunits. Then the strands are connected to form a continuous glycan molecule that encompasses the cell. Wherever their proximity allows it, the tetrapeptide chains that project from the glycan backbone can be cross-linked by an interpeptide bond between a free amino group on DAP and a free carboxy group on a nearby D-ala. The assembly of peptidoglycan on the outside of the plasma membrane is mediated by a group of periplasmic enzymes, which are transglycosylases, transpeptidases and carboxypeptidases. The mechanism of action of penicillin and related beta-lactam antibiotics is to block transpeptidase and carboxypeptidase enzymes during their assembly of the murein cell wall. Hence, the beta lactam antibiotics are said to "block cell wall synthesis" in the bacteria.

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 the phagocytic lysosome. The function of lysozyme is to lyse bacterial cells as a constitutive 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) , such that the muramic acid subunit shown in Figure 16(a) is the result of the action of lysozyme on bacterial peptidoglycan.

In Gram-positive bacteria there are numerous different peptide arrangements among peptidoglycans. The best studied is the murein of Staphylococcus aureus shown in Figure 17 below. In place of DAP (in E. coli) is the diamino acid, L-lysine (L-lys), and in place of the interpeptide bond (in Gram-negatives) is an interpeptide bridge of amino acids that connects a free amino group on lysine to a free carboxy group on D-ala of a nearby tetrapeptide side chain. This arrangement apparently allows for more frequent cross-bonding between nearby tetrapeptide side chains. In S. aureus, the interpeptide bridge is a peptide consisting of 5 glycine molecules (called a pentaglycine bridge). 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. In Gram-positive bacteria, peptidoglycans may vary in the amino acid in place of DAP or L-lys in position 3 of the tetrapeptide, and in the exact composition of the interpeptide bridge. At least eight different types of peptidoglycan exist in Gram-positive bacteria.


Figure 17. 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-glutamic acid; L-lys = L-lysine. This is one type of murein found in Gram-positive bacteria. Compared to the E. coli peptidoglycan (Figure 7) there is L-lys in place of DAP (diaminopimelic acid) in the tetrapeptide. The free amino group of L-lys is substituted with a glycine pentapeptide (gly-gly-gly-gly-gly-) which then becomes an interpeptide bridge forming a link with a carboxy group from D-ala in an adjacent tetrapeptide side chain. Gram-positive peptidoglycans differ from species to species, mainly in regards to the amino acids in the third position of the tetrapeptide side chain and in the amino acid composition of the interpeptide bridge.

Gram-negative bacteria may contain a single monomolecular layer of murein in their cell walls while Gram-positive bacteria are thought to have several layers or "wraps" of peptidoglycan. Closely associated with the layers of peptidoglycan in Gram-positive bacteria are a group of molecules called teichoic acids. Teichoic acids are linear polymers of polyglycerol or polyribitol substituted with phosphates and a few amino acids and sugars. The teichoic acid polymers are occasionally anchored to the plasma membrane (called lipoteichoic acid, LTA) apparently directed outward at right angles to the layers of peptidoglycan. The functions of teichoic acid are not known. They are essential to viability of Gram-positive bacteria in the wild. One idea is that they provide a channel of regularly-oriented negative charges for threading positively charged substances through the complicated peptidoglycan network. Another theory is that teichoic acids are in some way involved in the regulation and assembly of muramic acid subunits on the outside of the plasma membrane. There are instances, particularly in the streptococci, wherein teichoic acids have been implicated in the adherence of the bacteria to tissue surfaces.

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 18 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 is a lipid bilayer intercalated with proteins, superficially resembling the plasma membrane. The inner face of the outer membrane is composed of phospholipids similar to the phosphoglycerides that compose the plasma membrane. The outer face of the outer membrane may contain some phospholipid, but mainly it is formed by a different type of amphiphilic molecule which is composed of lipopolysaccharide (LPS). Outer membrane proteins usually traverse the membrane and in one case, anchor the outer membrane to the underlying peptidoglycan sheet.


Figure 18. Schematic illustration of the outer membrane, cell wall and plasma membrane of a Gram-negative bacterium. Note the structure and arrangement of molecules that constitute the outer membrane.

The LPS molecule that constitutes the outer face of the outer membrane is composed of a hydrophobic region, called Lipid A, that is attached to a hydrophilic linear polysaccharide region, consisting of the core polysaccharide and the O-specific polysaccharide.


Figure 19. Structure of LPS

The Lipid A head of the molecule inserts into the interior of the membrane, and the polysaccharide tail of the molecule faces the aqueous environment. Where the tail of the molecule inserts into the head there is an accumulation of negative charges such that a magnesium cation is chelated between adjacent LPS molecules. This provides the lateral stability for the outer membrane, and explains why treatment of Gram-negative bacteria with a powerful chelating agent, such as EDTA, causes dispersion of LPS molecules.

Bacterial lipopolysaccharides are toxic to animals. When injected in small amounts LPS or endotoxin activates macrophages to produce pyrogens, activates the complement cascade causing inflammation, and activates blood factors resulting in intravascular coagulation and hemorrhage. 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 ligands for bacterial attachment and confer some resistance to phagocytosis. Variation in the exact sugar content of the O polysaccharide (also referred to as the O antigen) accounts for multiple antigenic types (serotypes) among Gram-negative bacterial pathogens. Therefore. even though Lipid A is the toxic component in LPS, the polysaccharides nonetheless contribute to virulence of Gram-negative bacteria.

The proteins in the outer membrane of Escherichia coli are well characterized (see Table 5). About 400,00 copies of the Braun lipoprotein are covalently attached to the peptidoglycan sheet at one end and inserted into the hydrophobic interior of the membrane at the opposite end. A group of trimeric proteins called porins form pores of a fixed diameter through the lipid bilayer of the membrane. The omp C and omp F porins of E. coli are designed to allow passage of hydrophilic molecules up to mw of about 750 daltons. Larger molecules or harmful hydrophobic compounds (such as bile salts in the intestinal tract) are excluded from entry. Porins are designed in Gram-negative bacteria to allow passage of useful molecules (nutrients) through the barrier of the outer membrane, but to exclude passage harmful substances from the environment. The ubiquitous omp A protein in the outer membrane of E. coli has a porin like structure, and may function in uptake of specific ions, but it is also a receptor for the F pilus and an attachment site for bacterial viruses.

Table 5. Functions of the outer membrane components of Escherichia coli.
Component Function
Lipopolysaccharide (LPS) Permeability barrier
Mg++ bridges Stabilizes LPS and is essential for its permeability characteristics
Braun lipoprotein Anchors the outer membrane to peptidoglycan (murein) sheet
Omp C and Omp F porins proteins that form pores or channels through outer membrane for passage of hydrophilic molecules
Omp A protein  provides receptor for some viruses and bacteriocins; stabilizes mating cells during conjugation

S-layers

S-layer proteins form the outermost cell envelope component of a broad spectrum of bacteria and archaea.  S-layers are composed of a single protein or glycoprotein species (Mw 40-200 kDa) and exhibit either oblique, square or hexagonal lattice symmetry with unit cell dimensions in the range of 3 to 30 nm. S-layers are generally 5 to 10 nm thick and show pores of identical size (diameter, 2 - 8 nm) and morphology.

Crystalline bacterial cell surface layer (S-layer) proteins have been optimized during billions of years of biological evolution as constituent elements of one of the simplest self-assembly systems in nature. Isolated S-layer proteins have the intrinsic property to recrystallize into two-dimensional arrays on a broad spectrum of surfaces including silicon, metals and polymers, and to interfaces such as planar lipid films and liposomes. The well defined arrangement of functional groups on S-layer lattices allows the binding of molecules and particles in defined regular arrays. S-layers also represent templates for the formation of inorganic nanocrystal superlattices composed of CdS, Au, Ni, Pt, or Pd.

The self-assembly of S-layers illustrates a basic building principle in nature for generating large arrays of biomolecules with well-defined geometrical and physicochemical surface properties.

Many Gram-negative and Gram-positive bacteria, as well a many archaea possess a regularly structured layer called an S-layer attached to the outermost portion of their cell wall. It is composed of protein or glycoprotein and in electron micrographs, has a pattern resembling a tiled surface. Transmission electron micrograph of a freeze-etched, metal shadowed preparation of a bacterial cell with an S-layer with hexagonal lattice symmetry. Bar = 100nm.
http://www.foresight.org/conference/MNT7/Papers/Pum/index.html

S-layers have been associated with a number of possible functions. The S-layer may protect bacteria from harmful enzymes or changes in pH. It may contribute to virulence by protecting the bacterium against complement attack and phagocytosis. It is thought to protect E. coli from attack by the predatory bacterium, Bdellovibrio.

The S-layer can function as an adhesin, enabling the bacterium to adhere to host cell membranes and environmental surfaces in order to colonize. Many of the cell-associated protein adhesins used by pathogens are components of the S-layer.

A correlation between Gram stain reaction and cell wall properties of bacteria is summarized in Table 6. 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 6. Correlation of Grams stain with other properties of Bacteria.
Property  Gram-positive Gram-negative
Thickness of wall  thick (20-80 nm) thin (10 nm)
Number of layers  1  2
Peptidoglycan (murein) content  >50% 10-20%
Teichoic acids in wall present absent
Lipid and lipoprotein content  0-3% 58%
Protein content  0 9%
Lipopolysaccharide content  0  13%
Sensitivity to Penicillin G  yes no (1)
Sensitivity to lysozyme yes no (2)
(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. 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 bacteria can revert to cell wall-less forms (called spheroplasts or protoplasts) 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 Plasma Membrane

The plasma membrane, also called the cytoplasmic membrane, is the most dynamic structure of a procaryotic cell. Its main function is a s a selective 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 a unit, separating it from the environment. The bacterial membrane allows passage of water and uncharged molecules up to mw of about 100 daltons, but does not allow passage of larger molecules or any charged substances except by means special membrane transport processes and transport systems.

Bacterial membranes are composed of 40 percent phospholipid and 60 percent protein. The phospholipids are amphoteric molecules with a polar hydrophilic glycerol "head" attached via an ester bond to two nonpolar hydrophobic fatty acid tails, which naturally form a bilayer in aqueous environments. Dispersed within the bilayer are various structural and enzymatic proteins which carry out most membrane functions. At one time, it was thought that the proteins were neatly organized along the inner and outer faces of the membrane and that this accounted for the double track appearance of the membrane in electron micrographs. However, it is now known that while some membrane proteins are located and function on one side or another of the membrane, most proteins are partly inserted into the membrane, or possibly even traverse the membrane as channels from the outside to the inside. It is possible that proteins can move laterally along a surface of the membrane, but it is thermodynamically unlikely that proteins can be rotated within a membrane, which discounts early theories of how transport systems might work. The arrangement of proteins and lipids to form a membrane is called the fluid mosaic model, and is illustrated in Figure 20.


Figure 20. 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 proteins, which may be structural or functional, may be permanently or transiently associated with one side or the other of the membrane, or even be permanently built into the bilayer, while other proteins span the bilayer and may form transport channels through the membrane.

The membranes of Bacteria are structurally similar to the cell membranes of eucaryotes, except that bacterial membranes consist of saturated or monounsaturated fatty acids (rarely, polyunsaturated fatty acids) and do not normally contain sterols. The membranes of Archaea form bilayers functionally equivalent to bacterial membranes, but archaeal lipids are saturated, branched, repeating isoprenoid subunits that attach to glycerol via an ether linkage as opposed to the ester linkage found in glycerides of eukaryotic and bacterial membrane lipids (Figure 21). The structure of archaeal membranes is thought to be an adaptation to their existence and survival in extreme environments.


Figure 21. Generalized structure of a membrane lipids. (top). A phospholipid in the membrane of the bacterium Escherichia coli. The R1 and R2 positions on glycerol are substituted with saturated or monounsaturated fatty acids, with ester linkages to the glyceride. The R3 position is substituted with phosphatidylethanolamine, the most common substituent in this position in Bacteria. (bottom). An Archaeal membrane lipid. In contrast to bacterial phospholipids, which are glycerol esters of fatty acids, the lipids in membranes of Archaea are diethers of glycerol and long-chain, branched, saturated hydrocarbons called isoprenoids or which are made up of repeating C5 subunits. One of the major isoprenoids is the C20 molecule phytanol. The R3 position of glycerol may or may not be substituted. The structure of archaeal membrane lipids is thought to be an adaptation to extreme environments such as hot and acidic conditions where Archaea prevail in nature.

Functions of the Cytoplasmic Membrane

Since procaryotes lack any intracellular organelles for processes such as respiration or photosynthesis or secretion, the plasma membrane subsumes these processes for the cell and consequently has a variety of functions in energy generation, and biosynthesis. For example, the electron transport system that couples aerobic respiration and ATP synthesis is found in the procaryotic membrane. The photosynthetic chromophores that harvest light energy for conversion into chemical energy are located in the membrane. Hence, the plasma membrane is the site of oxidative phosphorylation and photophosphorylation in procaryotes, analogous to the functions of mitochondria and chloroplasts in eukaryotic cells. Besides transport proteins that selectively mediate the passage of substances into and out of the cell, procaryotic membranes may contain sensing proteins that measure concentrations of molecules in the environment or binding proteins that translocate signals to genetic and metabolic machinery in the cytoplasm. Membranes also contain 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 procaryotic membranes are listed in Table 7 and discussed below.

Table 7. Functions of the procaryotic plasma 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 proteins that mediate the passage of solutes through membranes are referred to variously as transport systems, carrier proteins, porters, and permeases. Transport systems operate by one of three transport processes as described below in Figure 22. In a uniport process, a solute passes through the membrane unidirectionally. In symport processes (also called cotransport) two solutes must be transported in the same direction at the same time; in antiport processes ( also called exchange diffusion), one solute is transported in one direction simultaneously as a second solute is transported in the opposite direction.

Figure 22. Transport processes in bacterial cells. Solutes enter or exit from bacterial cells by means of one of three processes: uniport, symport (also called cotransport) and antiport (also called exchange diffusion). Transport systems (Figure 13 below) operate by one or another of these processes.

Types of Transport Systems

Bacteria have a variety of types of transport systems which can be used alternatively in various environmental situations. The elaborate development of transport processes and transport systems in procaryotes probably reflects their need to concentrate substances inside the cytoplasm against the concentration (gradient) of the environment. Concentration of solutes in the cytoplasm requires the operation of an active transport system, of which there are two types in bacteria: ion driven transport systems (IDT) and binding-protein dependent transport systems (BPDT). The definitive feature of an active transport system is the accumulation of the solute in the cytoplasm at concentrations far in excess of the environment. According to the laws of physical chemistry, this type of process requires energy.
 


Figure 23. Operation of bacterial transport systems. Bacterial transport systems are operated by transport proteins (sometimes called carriers, porters or permeases) in the plasma membrane. Facilitated diffusion is a carrier-mediated system that does not require energy and does not concentrate solutes against a gradient. Active transport systems such as Ion-driven transport and Binding protein-dependent transport, use energy and concentrate molecules against a concentration gradient. Group translocation systems, such as the phosphotransferase (pts) system in Escherichia coli, use energy during transport and modify the solute during its passage across the membrane.

There are four types of carrier-mediated transport systems in procaryotes. The carrier is a protein (or group of proteins) that functions in the passage of a small molecule from one side of a membrane to the other side. A transport system may be a single transmembranous protein that forms a channel that admits passage of a specific solute, or it may be a coordinated system of proteins that binds and sequentially passes a small molecule through the membrane. Transport systems have the property of specificity for the solute transported. Some transport systems transport a single solute with the same specificity and kinetics as an enzyme. Some transport systems will transport (structurally) related molecules, although at reduced efficiency compared to their primary substrate. Most transport systems transport specific sugars, amino acids, anions or cations that are of nutritional value to the bacterium.

Facilitated diffusion systems (FD) are the least common type of transport system in bacteria. Actually, the glycerol uniporter in E. coli is the only well known facilitated diffusion system. FD involves the passage of a specific solute through a carrier that forms a channel in the membrane. The solute can move in either direction through the membrane to the point of of equilibrium on both sides of the membrane. Although the system is carrier-mediated and specific, no energy is expended in the transport process. For this reason the glycerol molecule cannot be accumulated against the concentration gradient.

Ion driven transport systems (IDT) and Binding-protein dependent transport systems (BPDT) are active transport systems that are used for transport of most solutes by bacterial cells. IDT is used for accumulation of many ions and amino acids; BPDT is frequently used for sugars and amino acids. IDT is a symport or antiport process that uses a hydrogen ion (H+) i.e., proton motive force (pmf), or some other cation, i.e., chemiosmotic potential, to drive the transport process. IDT systems such as the lactose permease of E. coli utilize the consumption of a hydrogen ion during the transport of lactose. Thus the energy expended during active transport of lactose is in the form of pmf. The lactose permease is a single transmembranous polypeptide that spans the membrane seven times forming a channel that specifically admits lactose.

Binding-protein dependent transport systems (BPDT), such as the histadine transport system in E. coli, are composed of four proteins. Two proteins form a membrane channel that allows passage of the histadine. A third protein resides in the periplasmic space where it is able to bind the amino acid and pass it to a forth protein which admits the amino acid into the membrane channel. Driving the solute through the channel involves the expenditure of energy, which is provided by the hydrolysis of ATP.

Group translocation systems (GT), more commonly known as the phosphotransferase system (PTS) in E. coli, are used primarily for the transport of sugars. Like binding protein-dependent transport systems, they are composed of several distinct components. However, GT systems specific for one sugar may share some of their components with other group transport systems. In E. coli, glucose may be transported by a group translocation process that involves the phosphotransferase system. The actual carrier in the membrane is a protein channel fairly specific for glucose. Glucose specifically enters the channel from the outside, but in order to exit into the cytoplasm, it must first be phosphorylated by the phosphotransferase system. The PTS derives energy from the metabolic intermediate phosphoenol pyruvate (PEP). PEP is hydrolyzed to pyruvate and glucose is phosphorylated to form glucose-phosphate during the process. Thus, by the expenditure of a single molecule of high energy phosphate, glucose is transported and changed to glucose-phosphate.

Table 8. Distinguishing characteristics of bacterial transport systems
PD = passive diffusion
FD = facilitated diffusion
IDT = ion-driven transport
BPDT = binding protein dependent transport
GT = group translocation
Property  PD FD IDT BPDT GT
carrier mediated  - + + + +
conc. against gradient  - - + + NA
specificity  - + + + +
energy expended  - - pmf  ATP PEP
solute modified during transport  - - - - +

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 photophorylation.

Figure 24 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 24. Schematic view of the plasma membrane of Escherichia coli. The S and M rings which constitute the flagellar motor are shown. The motor ring is imbedded in the phospholipid bilayer. It is powered by pmf 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. Several other transmembranous proteins are transport systems which are operating by either symport or antiport processes.

The plasma membrane of procaryotes may invaginate into the cytoplasm or form stacks or vesicles attached to the inner membrane surface. These structures are sometimes referred to as mesosomes. Such internal membrane systems may be analogous to the cristae of mitochondria or the thylakoids of chloroplasts which increase the surface area of membranes to which enzymes are bound for specific enzymatic functions. The photosynthetic apparatus (light harvesting pigments and ATPase) of photosynthetic procaryotes is contained in these types of membranous structures. Mesosomes may also represent specialized membrane regions involved in DNA replication and segregation, cell wall synthesis, or increased enzymatic activity. Membrane foldings and vesicles sometimes appear in electron micrographs of procaryotic cells as artifacts of preparative techniques. These membranous structures, of course, are not mesosomes, but their existence does not prove that mesosomes are not present in procaryotes, and there are several examples of procaryotic membrane topology and appearance that are suggestive of mesosomes.

There are a few antibiotics (e.g. polymyxin), hydrophobic agents (e.g. bile salts), and proteins (e.g. complement) that can damage bacterial membranes.

The Periplasm

Between the inner (plasma) and outer membranes of Gram-negative bacteria and spirochetes is a space called the periplasm or periplasmic space (See Figures 9 and 18). Actually, the peptidoglycan sheet resides within the periplasm. The periplasm is a very active compartment of the cell, containing enzymes for assembly of cell wall and membrane components, various degradative or detoxifying enzymes, secretion systems, sensing proteins for chemotaxis and signal transduction, and binding proteins for solutes taken up by BPDT transport systems. Components of the periplasm are needed in this region of the cell and are bounded or "trapped" by the two membranes of the cell. In the case of spirochetes, their flagella (called endoflagella or periplasmic flagella) rotate within the periplasm and impart the flexing and screw-like rotation characteristic of spirochete motility.
Table 9. Representative periplasmic proteins in E. coli.

The Cytoplasm

The cytoplasm of bacterial cells consists consists of an aqueous solution of three groups of molecules: macromolecules such as proteins (enzymes), mRNA and tRNA;  small molecules that are energy sources, precursors of macromolecules, metabolites or vitamins;  and various inorganic ions and cofactors (see Tables 9, 10, 11).  The primary structural components found in the cytoplasm are the nucleoid and ribosomes, and possibly some type of inclusion. 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 9. Molecular composition of E. coli under conditions of balanced growth. Percentage of  dry weight refers to all structural and  cytoplasmic components.
Molecule Percentage of dry weight
Protein 

Total RNA 

DNA 

Phospholipid

Lipopolysaccharide 

Murein 

Glycogen 

Small molecules: precursors, metabolites, vitamins, etc.

Inorganic ions 

Total dry weight 

 55

20.5

3.1 

9.1 

3.4 

2.5 

2.5 

2.9

1.0

100.0
 
Table 10. Small molecules present in the cytoplasm of a growing bacterial cell.
Molecule Approximate number of kinds
Amino acids, their precursors and derivatives 

Nucleotides, their precursors and derivatives 

Fatty acids and their precursors 

Sugars, carbohydrates and their precursors or derivatives

quinones, porphyrins, vitamins, coenzymes and prosthetic groups and their precursors 

 120

100

50

250

300

Table 11. Inorganic ions present in the cytoplasm of a growing bacterial cell. 
Ion  Function
K+ Maintenance of ionic strength; cofactor for certain enzymes
NH4+ Principal form of inorganic N for assimilation
Ca++ Cofactor for certain enzymes
Fe++ Present in cytochromes and other metalloenzymes
Mg++ Cofactor for many enzymes; stabilization of outer membrane of Gram-negative bacteria
Mn++ Present in certain metalloenzymes
Co++ Trace element constituent of vitamin B12 and its coenzyme derivatives and found in certain metalloenzymes
Cu++ Trace element present in certain metalloenzymes
Mo++ Trace element present in certain metalloenzymes
Ni++ Trace element present in certain metalloenzymes
Zn++ Trace element present in certain metalloenzymes
SO4-- Principal form of inorganic S for assimilation
PO4---  Principal form of P for assimilation and a participant in many metabolic reactions

The bacterial chromosome (nucleoid) is typically one large circular molecule of DNA, more or less free in the cytoplasm, although coiled and supercoiled and anchored by proteins. 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 a semiconservative fashion to make an exact copy of the molecule for distribution to progeny cells. However, the eucaryotic processes of meiosis and mitosis are absent in procaryotes. Replication and segregation of procaryotic DNA is coordinated by the membrane and various proteins in the cytoplasm.

 
Figure 25. 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. Ribosomes are composed of proteins and RNA. 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. The 70S ribosomes that occur in eucaryotic mitochondria and chloroplasts contain ssrRNA closely related to bacterial ribosomal RNA. his is taken as a major line of evidence that these organelles are descended from procaryotes.

 
Figure 26. 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 12. Some inclusions in bacterial cells.
Cytoplasmic inclusions Where found Composition Function
glycogen many bacteria e.g. E. coli polyglucose reserve carbon and energy source
polybetahydroxybutyric acid (PHB) many bacteria e.g. Pseudomonas polymerized hydroxy butyrate reserve carbon and energy source
polyphosphate (volutin granules) many bacteria e.g. Corynebacterium linear or cyclical polymers of PO4 reserve phosphate; possibly a reserve of high energy phosphate
sulfur globules phototrophic purple and green sulfur bacteria and lithotrophic colorless sulfur bacteria elemental sulfur reserve of electrons (reducing source) in phototrophs; reserve energy source in lithotrophs
gas vesicles aquatic bacteria especially cyanobacteria protein hulls or shells inflated with gases buoyancy (floatation) in the vertical water column
parasporal crystals endospore-forming bacilli (genus Bacillus) protein unknown but toxic to certain insects
magnetosomes certain aquatic bacteria magnetite (iron oxide) Fe3O4  orienting and migrating along geo- magnetic field lines
carboxysomes many autotrophic bacteria enzymes for autotrophic CO2 fixation site of CO2 fixation
phycobilisomes cyanobacteria phycobiliproteins light-harvesting pigments
chlorosomes Green bacteria lipid and protein and bacteriochlorophyll light-harvesting pigments and antennae


Figure 27. 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

Endospores

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 28. 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 13. Differences between endospores and vegetative cells
Property Vegetative cells Endospores
Surface coats Typical Gram-positive murein cell wall polymer Thick spore coat, cortex, and peptidoglycan core wall
Microscopic appearance Nonrefractile Refractile
Calcium dipicolinic acid Absent Present in core
Cytoplasmic water activity High Very low
Enzymatic activity Present Absent
Macromolecular synthesis Present Absent
Heat resistance Low High
Resistance to chemicals and acids Low High
Radiation resistance Low High
Sensitivity to lysozyme Sensitive Resistant
Sensitivity to dyes and staining Sensitive Resistant


Figure 29. 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 30. 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.

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Written and edited by Kenneth Todar  University of Wisconsin-Madison  Department of Bacteriology  All rights reserved