Chapter 2: Structure of Bacterial Cells

Bacteria are classified by shape into three basic groups: cocci, bacilli, and spirochetes (Figure 2–1). The cocci are round, the bacilli are rods, and the spirochetes are spiral-shaped. Some bacteria are variable in shape and are said to be pleomorphic (heterogeneous shape). The shape of a bacterium is determined by its rigid cell wall. The microscopic appearance of a bacterium is one of the most important criteria used in its identification.

In addition to their characteristic shapes, the arrangement of bacteria is important. For example, certain cocci occur in pairs (diplococci), some in chains (streptococci), and others in grapelike clusters (staphylococci). These arrangements are determined by the orientation and degree of attachment of the bacteria at the time of cell division. The arrangement of rods and spirochetes is medically less important and is not described in this introductory chapter.

Bacteria range in size from about 0.2 to 5 µm (Figure 2–2). The smallest bacteria (Mycoplasma) are about the same size as the largest viruses (poxviruses) and are the smallest organisms capable of existing outside a host. The longest bacteria are the size of some yeasts and human red blood cells (7 µm).

The structure of a typical bacterium is illustrated in Figure 2–3, and the important features of each component are presented in Table 2–1.

Cell Wall

The cell wall is the outermost component common to all bacteria (except Mycoplasma species, which are bounded by a cell membrane, not a cell wall). Some bacteria have surface features external to the cell wall, such as capsule, flagella, and pili, which are less common components and are discussed next.

The cell wall is located external to the cytoplasmic membrane and is composed of peptidoglycan. The peptidoglycan provides structural support and maintains the characteristic shape of the cell.

Cell Walls of Gram-Positive and Gram-Negative Bacteria

The structure, chemical composition, and thickness of the cell wall differ in gram-positive and gram-negative bacteria (Table 2–2, Figure 2–4A, and “Gram Stain” box).

1. The peptidoglycan layer is much thicker in gram-positive than in gram-negative bacteria. Many gram-positive bacteria also have fibers of teichoic acid that protrude outside the peptidoglycan, whereas gram-negative bacteria do not have teichoic acids.

2. In contrast, the gram-negative bacteria have a complex outer layer consisting of lipopolysaccharide (LPS), lipoprotein, and phospholipid. Together with the cell wall, this gram-negative architecture is referred to as the “envelope.” Lying between the outer-membrane layer and the cytoplasmic membrane in gram-negative bacteria is the periplasmic space, which is the site, in some species, of enzymes called β-lactamases that degrade penicillins and other β-lactam drugs.

The cell wall in gram-positive organisms or cell envelope in gram-negative organisms has several other important properties:

1. In gram-negative bacteria, the envelope contains endotoxin, an LPS (Lipopolysaccharide and Endotoxins).

2. Both gram-positive and gram-negative bacteria contain polysaccharides and proteins on their surface that are antigens useful in laboratory identification.

3. Porin proteins play a role in facilitating the passage of small, hydrophilic molecules into the cell. Several types of porin proteins can be found in the outer membrane of gram-negative bacteria, allowing the entry of essential substances such as sugars, amino acids, vitamins, and metals as well as many antimicrobial drugs such as penicillins. Porins have also been identified in gram-positive bacteria, where they are anchored to the cell wall.

Cell Walls of Acid-Fast Bacteria

Mycobacteria (e.g., Mycobacterium tuberculosis) have an unusual cell wall, resulting in their inability to be Gram-stained (Figure 2–4B). These bacteria are said to be acid-fast because they resist decolorization with acid–alcohol after being stained with carbolfuchsin. This property is related to the high concentration of lipids, called mycolic acids, in the cell wall of mycobacteria.

Note that Nocardia asteroides, a bacterium that can cause lung and brain infections in immunocompromised individuals, is weakly acid-fast. The meaning of the term “weakly” is that if the acid-fast staining process uses a weaker solution of hydrochloric acid to decolorize than that used in the stain for mycobacteria, then N. asteroides will not decolorize. However, if the regular-strength hydrochloric acid is used, N. asteroides will decolorize.

In view of their importance, three components of the cell wall (i.e., peptidoglycan, LPS, and teichoic acid) are discussed in detail here.

Peptidoglycan

Peptidoglycan is a complex, interwoven network that surrounds the entire cell and is composed of a single covalently linked macromolecule. It is found only in bacterial cell walls. It provides rigid support for the cell, is important in maintaining the characteristic shape of the cell, and allows the cell to withstand low osmotic pressure. A representative segment of the peptidoglycan layer is shown in Figure 2–5. The term peptidoglycan is derived from the peptides and the sugars (glycan) that make up the molecule. Synonyms for peptidoglycan are murein and mucopeptide.

Figure 2–5 illustrates the carbohydrate backbone, which is composed of alternating N-acetylmuramic acid and N-acetylglucosamine molecules. Attached to each of the muramic acid molecules is a tetrapeptide consisting of both D- and L-amino acids, the precise composition of which differs from one bacterium to another. Two of these amino acids are worthy of special mention: diaminopimelic acid, which is unique to bacterial cell walls, and D-alanine, which is involved in the cross-links between the tetrapeptides and in the action of penicillin. Note that this tetrapeptide contains the rare D-isomers of amino acids; most proteins contain the L-isomer. The other important component in this network is the peptide cross-link between the two tetrapeptides. The cross-links vary among species; in Staphylococcus aureus, for example, five glycines link the terminal D-alanine to the penultimate L-lysine.

Because peptidoglycan is present in bacteria but not in human cells, it is a good target for antibacterial drugs. Several of these drugs, such as penicillins, cephalosporins, and vancomycin, inhibit the synthesis of peptidoglycan by inhibiting the transpeptidase that makes the cross-links between the two adjacent tetrapeptides (see Chapter 10).

Lysozyme, an enzyme present in human tears, mucus, and saliva, can cleave the peptidoglycan backbone by breaking its glycosyl bonds, thereby contributing to the natural resistance of the host to microbial infection. Lysozyme-treated bacteria may swell and rupture as a result of the entry of water into the cells, which have a high internal osmotic pressure. However, if the lysozyme-treated cells are in a solution with the same osmotic pressure as that of the bacterial interior, they will survive as spherical forms, called protoplasts, surrounded only by a cytoplasmic membrane.

Lipopolysaccharide

The LPS of the outer membrane of the cell wall of gram-negative bacteria is endotoxin. It is responsible for many of the features of disease, such as fever and shock (especially hypotension), caused by these organisms (see Endotoxins). It is called endotoxin because it is an integral part of the cell envelope, in contrast to exotoxins, which are actively secreted from the bacteria. The constellation of symptoms caused by the endotoxin of one gram-negative bacterium is similar to another, but the severity of the symptoms can differ greatly. In contrast, the symptoms caused by exotoxins of different bacteria are usually quite different.

The LPS is composed of three distinct units (Figure 2–6):

1. A phospholipid called lipid A, which is responsible for the toxic effects.

2. A core polysaccharide of five sugars linked through ketodeoxyoctulonate (KDO) to lipid A.

3. An outer polysaccharide consisting of up to 25 repeating units of three to five sugars. This outer polymer is the important somatic, or O, antigen of several gram-negative bacteria that is used to identify certain organisms in the clinical laboratory. Some bacteria, notably members of the genus Neisseria, have an outer lipooligosaccharide (LOS) containing very few repeating units of sugars.

Teichoic Acid

Teichoic acids are fibers located in the outer layer of the gram-positive cell wall and extend from it. They are composed of polymers of either glycerol phosphate or ribitol phosphate. Some polymers of glycerol teichoic acid penetrate the peptidoglycan layer and are covalently linked to the lipid in the cytoplasmic membrane, in which case they are called lipoteichoic acid; others anchor to the muramic acid of the peptidoglycan.

The medical importance of teichoic acids lies in their ability to induce inflammation and septic shock when caused by certain gram-positive bacteria; that is, they activate the same pathways as does endotoxin (LPS) in gram-negative bacteria. Teichoic acids also mediate the attachment of staphylococci to mucosal cells. Gram-negative bacteria do not have teichoic acids.

Cytoplasmic Membrane

Just inside the peptidoglycan layer of the cell wall lies the cytoplasmic membrane, which is composed of a phospholipid bilayer similar in microscopic appearance to that in eukaryotic cells. They are chemically similar, but eukaryotic membranes contain sterols, whereas prokaryotes generally do not. The only prokaryotes that have sterols in their membranes are members of the genus Mycoplasma. The membrane has four important functions: (1) active transport of molecules into the cell, (2) energy generation by oxidative phosphorylation, (3) synthesis of precursors of the cell wall, and (4) secretion of enzymes and toxins.

Cytoplasm

The cytoplasm has two distinct areas when seen in the electron microscope:

1. An amorphous matrix that contains ribosomes, nutrient granules, metabolites, and plasmids.

2. An inner, nucleoid region composed of DNA.

Ribosomes

Bacterial ribosomes are the site of protein synthesis as in eukaryotic cells, but they differ from eukaryotic ribosomes in size and chemical composition. Bacterial ribosomes are 70S in size, with 50S and 30S subunits, whereas eukaryotic ribosomes are 80S in size, with 60S and 40S subunits. The differences in both the ribosomal RNAs and proteins constitute the basis of the selective action of several antibiotics that inhibit bacterial, but not human, protein synthesis (see Chapter 10).

Granules

The cytoplasm contains several different types of granules that serve as storage areas for nutrients and stain characteristically with certain dyes. For example, volutin is a reserve of high energy stored in the form of polymerized metaphosphate. It appears as a “metachromatic” granule since it stains red with methylene blue dye instead of blue as one would expect. Metachromatic granules are a characteristic feature of Corynebacterium diphtheriae, the cause of diphtheria.

Nucleoid

The nucleoid is the area of the cytoplasm in which DNA is located. The DNA of most prokaryotes is a single, circular molecule; however, there are important exceptions. For instance, the genome of Vibrio cholerae, the causative agent of cholera, is composed of two circular chromosomes. Borrelia burgdorferi, the spirochete that causes Lyme disease, is composed of a linear chromosome and multiple circular and linear plasmids (see below). The size of bacterial genomes varies widely, with the smallest genome containing just over 130 genes and the largest containing approximately 11,600 genes. By contrast, human DNA has approximately 25,000 genes.

Because the bacterial nucleoid contains no nuclear membrane, no nucleolus, no mitotic spindle, and no histones, there is little resemblance to the eukaryotic nucleus. One major difference between bacterial DNA and eukaryotic DNA is that bacterial DNA has no introns, whereas eukaryotic DNA does.

Plasmids

Plasmids are extrachromosomal, double-stranded, circular DNA molecules that are capable of replicating independently of the bacterial chromosome. Although plasmids are usually extrachromosomal, they can be integrated into the bacterial chromosome. Plasmids occur in both gram-positive and gram-negative bacteria, and several different types of plasmids can exist in one cell:

1. Transmissible plasmids can be transferred from cell to cell by conjugation (see Chapter 4 for a discussion of conjugation). They are large (molecular weight [MW] 40–100 million), since they contain about a dozen genes responsible for synthesis of the sex pilus and for the enzymes required for transfer. They are usually present in a few (1–3) copies per cell.

2. Nontransmissible plasmids are small (MW 3–20 million) since they do not contain the transfer genes; they are frequently present in many (10–60) copies per cell.

Plasmids carry the genes for the following functions and structures of medical importance:

1. Antibiotic resistance, which is mediated by a variety of enzymes, such as the β-lactamase of S. aureus, Escherichia coli, and Klebsiella pneumoniae.

2. Exotoxins, such as the enterotoxins of E. coli, anthrax toxin of Bacillus anthracis, exfoliative toxin of S. aureus, and tetanus toxin of Clostridium tetani.

3. Pili (fimbriae), which mediate the adherence of bacteria to epithelial cells.

4. Resistance to heavy metals, such as mercury, the active component of some antiseptics (e.g., Merthiolate and Mercurochrome), and silver, which is mediated by a reductase enzyme.

5. Resistance to ultraviolet light, which is mediated by DNA repair enzymes.

6. Bacteriocins, which are toxic proteins produced by certain bacteria that are lethal for other bacteria. Two common mechanisms of action of bacteriocins are (i) degradation of bacterial cell membranes by producing pores in the membrane and (ii) degradation of bacterial DNA by deoxyribonuclease (DNase). Examples of bacteriocins produced by medically important bacteria are colicins made by E. coli and pyocins made by Pseudomonas aeruginosa. Bacteria that produce bacteriocins have a selective advantage in the competition for food sources over those that do not. However, the medical importance of bacteriocins is that they may be useful in treating infections caused by antibiotic-resistant bacteria.

Transposons

Transposons are pieces of DNA that move readily from one site to another either within or between the DNAs of bacteria, plasmids, and bacteriophages. Because of their unusual ability to move, they are nicknamed “jumping genes.” Some transposons move by replicating their DNA and inserting the new copy into another site (replicative transposition), whereas others are excised from the site without replicating and then inserted into the new site (direct transposition). Transposons can code for drug-resistant enzymes, toxins, or a variety of metabolic enzymes and can either cause mutations in the gene into which they insert or alter the expression of nearby genes.

Transposons typically have four identifiable domains. On each end is a short DNA sequence of inverted repeats, which are involved in the integration of the transposon into the recipient DNA. The second domain is the gene for the transposase, which is the enzyme that mediates the excision and integration processes. The third region is the gene for the repressor that regulates the synthesis of both the transposase and the protein encoded by the fourth domain, which, in many cases, is an enzyme mediating antibiotic resistance (Figure 2–7). Note that for simplicity, the repressor gene is not shown in Figure 2–7.

Antibiotic resistance genes are transferred from one bacterium to another primarily by conjugation (see Chapter 4). This transfer is mediated primarily by plasmids, but some transposons, called conjugative transposons, are capable of transferring antibiotic resistance as well.

In contrast to plasmids or bacterial viruses, transposons are not capable of independent replication; they replicate as part of the DNA in which they are integrated. More than one transposon can be located in the DNA; for example, a plasmid can contain several transposons carrying drug-resistant genes. Insertion sequences are a type of transposon that have fewer bases (800–1500 base pairs), since they do not code for their own integration enzymes. They can cause mutations at their site of integration and can be found in multiple copies at the ends of larger transposon units.

Structures Outside the Cell Wall

Capsule

The capsule is a gelatinous layer covering the entire bacterium. It is typically composed of polysaccharide. The sugar components of the polysaccharide vary from one species of bacteria to another and frequently determine the serologic type (serotype) within a species. For example, there are 91 different serotypes of Streptococcus pneumoniae, which are distinguished by the antigenic differences of the sugars in the polysaccharide capsule.

The capsule is important for four reasons:

1. It is a determinant of virulence of many bacteria since it limits the ability of phagocytes to engulf the bacteria. Negative charges on the capsular polysaccharide repel the negatively charged cell membrane of the neutrophil and prevent it from ingesting the bacteria. Variants of encapsulated bacteria that have lost the ability to produce a capsule are usually nonpathogenic.

2. Specific identification of an organism can be made by using antiserum against the capsular polysaccharide. In the presence of the homologous antibody, the capsule will swell greatly. This swelling phenomenon, which is used in the clinical laboratory to identify certain organisms, is called the Quellung reaction.

3. Capsular polysaccharides are used as the antigens in certain vaccines because they are capable of eliciting protective antibodies. For example, the capsular polysaccharide of S. pneumoniae, Neisseria meningitidis, and Haemophilus influenzae is the immunogen in the current vaccine against these bacteria.

4. The capsule may play a role in the adherence of bacteria to human tissues, which is an important initial step in causing infection.

Flagella

Flagella are long, whip-like appendages that move the bacteria toward nutrients and other attractants, a process called chemotaxis. The long filament, which acts as a propeller, is composed of many subunits of a single protein, flagellin, arranged in several intertwined chains. The energy for movement, the proton motive force, is provided by adenosine triphosphate (ATP), derived from the passage of ions across the membrane.

Flagellated bacteria have a characteristic number and location of flagella: some bacteria have one, and others have many; in some, the flagella are located at one end, and in others, they are all over the outer surface. Only certain bacteria have flagella. Many rods do, but most cocci do not and are therefore nonmotile. Spirochetes move by using a flagellum-like structure called the axial filament, which wraps around the spiral-shaped cell to produce an undulating motion.

Flagella are medically important for two reasons:

1. Some species of motile bacteria (e.g., E. coli and Proteus species) are common causes of urinary tract infections. Flagella may play a role in pathogenesis by propelling the bacteria up the urethra into the bladder.

2. Some species of bacteria (e.g., Salmonella species) are identified in the clinical laboratory by the use of specific antibodies against flagellar proteins.

Pili (Fimbriae)

Pili are hairlike filaments that extend from the cell surface. They are shorter and straighter than flagella and are composed of subunits of pilin, a protein arranged in helical strands. They are found mainly on gram-negative organisms.

Pili have two important roles:

1. They mediate the attachment of bacteria to specific receptors on the human cell surface, which is a necessary step in the initiation of infection for some organisms. Mutants of Neisseria gonorrhoeae that do not form pili are nonpathogens.

2. A specialized kind of pilus, the sex pilus, forms the attachment between the donor and the recipient bacteria during conjugation (see Chapter 4).

Glycocalyx (Slime Layer)

The glycocalyx is a polysaccharide coating that is secreted by many bacteria. It covers surfaces like a film and allows the bacteria to adhere firmly to various structures (e.g., skin, heart valves, prosthetic joints, and catheters). The glycocalyx is an important component of biofilms (see Table 7–6). The medical importance of the glycocalyx is illustrated by the finding that it is the glycocalyx-producing strains of P. aeruginosa that cause respiratory tract infections in cystic fibrosis patients, and it is the glycocalyx-producing strains of Staphylococcus epidermidis and viridans streptococci that cause endocarditis. The glycocalyx also mediates adherence of certain bacteria to the surface of teeth. This plays an important role in the formation of plaque.

Bacterial Spores

These highly resistant structures are formed in response to adverse conditions by two genera of medically important gram-positive rods: the genus Bacillus, which includes the agent of anthrax, and the genus Clostridium, which includes the agents of tetanus and botulism. Spore formation (sporulation) occurs when nutrients, such as sources of carbon and nitrogen, are depleted (Figure 2–8). The spore forms inside the cell and contains bacterial DNA, a small amount of cytoplasm, cell membrane, peptidoglycan, very little water, and most importantly, a thick, keratin-like coat that is responsible for the remarkable resistance of the spore to heat, dehydration, radiation, and chemicals. This resistance may be mediated by dipicolinic acid, a calcium ion chelator found only in spores.

Once formed, the spore has no metabolic activity and can remain dormant for many years. Upon exposure to water and the appropriate nutrients, specific enzymes degrade the coat, water and nutrients enter, and germination into a potentially pathogenic bacterial cell occurs. Note that this differentiation process is not a means of reproduction since one cell produces one spore that germinates into one cell.

The medical importance of spores lies in their extraordinary resistance to heat and chemicals. As a result of their resistance to heat, sterilization cannot be achieved by boiling. Steam heating under pressure (autoclaving) at 121°C, for at least 15 minutes, is required to ensure the sterility of products for medical use. Spores are often not seen in clinical specimens recovered from patients infected by spore-forming organisms because the supply of nutrients is adequate.

Table 2–4 describes the medically important features of bacterial spores.