# Lecture 3: Prokaryotic Cell Structure
## Microbiology
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## Learning Objectives
By the end of this lecture, students will be able to:
1. Describe the general features that distinguish prokaryotic cells from eukaryotic cells
2. Explain the structure and function of the bacterial cell envelope, including the plasma membrane, cell wall, and outer membrane
3. Differentiate between Gram-positive and Gram-negative cell wall architecture
4. Describe the structure and function of external appendages (flagella, pili, fimbriae)
5. Explain the roles of capsules, endospores, and inclusion bodies
6. Identify the components of the bacterial cytoplasm, including the nucleoid and ribosomes
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## Lecture Content
### I. Overview of Prokaryotic Cells
Prokaryotes are defined by the absence of a membrane-bound nucleus and other membrane-bound organelles. They are generally smaller than eukaryotic cells, ranging from about 0.2 to 10 micrometers in length, and their high surface-area-to-volume ratio facilitates rapid nutrient exchange and fast growth.
Bacteria exhibit several common shapes. A **coccus** is spherical, as seen in *Staphylococcus* and *Streptococcus*. A **bacillus** is rod-shaped, exemplified by *Escherichia coli* and *Bacillus subtilis*. **Spirillum** describes a rigid spiral form (*Spirillum volutans*), while a **spirochete** is a flexible spiral that moves using internal endoflagella (*Treponema*, *Borrelia*). **Vibrio** refers to comma-shaped organisms like *Vibrio cholerae*, and a **coccobacillus** is a short rod, as seen in *Haemophilus influenzae*. Beyond individual cell shape, bacteria display characteristic arrangements: diplococci form pairs, streptococci align in chains, staphylococci cluster in grape-like aggregates, and less common groupings include tetrads and sarcinae (cubes of eight).
### II. The Plasma (Cytoplasmic) Membrane
The plasma membrane follows the fluid mosaic model, consisting of a phospholipid bilayer studded with embedded and peripheral proteins. In bacteria, the phospholipids contain ester-linked fatty acids, in contrast to the ether-linked isoprenoid chains found in archaea. Some bacteria incorporate hopanoids into their membranes, molecules that play a stabilizing role analogous to cholesterol in eukaryotic cells.
The plasma membrane serves many essential functions. It acts as a selective permeability barrier, controlling the movement of molecules into and out of the cell. Active transport systems, including ABC transporters and the phosphotransferase system (PTS) for sugars, allow the cell to accumulate nutrients against concentration gradients. In aerobic bacteria, the plasma membrane houses the electron transport chain and the machinery for oxidative phosphorylation. It is also the site of lipid and cell wall biosynthesis, contains the methyl-accepting chemotaxis proteins that sense chemical gradients, and provides the platform for the FtsZ ring that orchestrates cell division.
### III. The Cell Wall
The bacterial cell wall provides structural rigidity, protects the cell against osmotic lysis, and determines cell shape. Its defining structural polymer is **peptidoglycan (murein)**, a macromolecule unique to bacteria. Peptidoglycan consists of alternating residues of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) linked by beta-1,4-glycosidic bonds. Tetrapeptide side chains extend from the NAM residues, and cross-linking of these peptide chains provides the tensile strength that keeps the cell intact. In Gram-negative bacteria, cross-links form directly between diaminopimelic acid (DAP) on one chain and D-alanine on another. In many Gram-positive species, such as *S. aureus*, a pentaglycine bridge connects L-lysine to D-alanine. Peptidoglycan is the target of several important antibiotics: penicillins and cephalosporins inhibit transpeptidases (penicillin-binding proteins or PBPs), vancomycin binds the D-Ala-D-Ala terminus of the peptide side chains, and lysozyme cleaves the NAG-NAM glycosidic bond.
#### A. Gram-Positive Cell Wall
The Gram-positive cell wall features a thick peptidoglycan layer, 20 to 80 nm and composed of multiple layers. Embedded within this peptidoglycan are **teichoic acids**, polymers of glycerol phosphate or ribitol phosphate. **Wall teichoic acids (WTA)** are covalently linked to peptidoglycan, while **lipoteichoic acids (LTA)** are anchored in the plasma membrane and extend outward through the peptidoglycan. Together, teichoic acids help maintain cation homeostasis (particularly Mg2+ and Ca2+), regulate autolysins and cell division, serve as receptors for bacteriophages, and contribute to virulence. Gram-positive bacteria lack an outer membrane and either have no periplasmic space or only a very thin periplasm-like region. Representative organisms include *Staphylococcus aureus*, *Streptococcus pyogenes*, *Bacillus subtilis*, and *Clostridium* species.
#### B. Gram-Negative Cell Wall
The Gram-negative cell wall has a thin peptidoglycan layer, typically only 1 to 3 nm and often a single layer. External to the peptidoglycan sits the **outer membrane (OM)**, an asymmetric lipid bilayer whose inner leaflet is composed of phospholipids and whose outer leaflet is dominated by **lipopolysaccharide (LPS)**. LPS consists of three regions: **Lipid A**, the endotoxic moiety embedded in the membrane, which can trigger fever, inflammation, and septic shock when released; the **core polysaccharide**, a short sugar chain; and the **O-antigen (O-polysaccharide)**, repeating sugar units that extend outward and serve as a major surface antigen used in serotyping. The outer membrane also contains **porins**, trimeric beta-barrel proteins that form channels allowing passive diffusion of small hydrophilic molecules (generally less than about 600 Da), and **Braun's lipoprotein**, which tethers the outer membrane to the peptidoglycan. Between the inner and outer membranes lies the **periplasmic space**, a gel-like compartment containing degradative enzymes (such as beta-lactamases), binding proteins for nutrient transport, and chemoreceptors. Examples of Gram-negative bacteria include *Escherichia coli*, *Pseudomonas aeruginosa*, *Neisseria*, and *Salmonella*.
A side-by-side cross-sectional comparison of Gram-positive and Gram-negative bacterial cell envelopes. Left panel (Gram-positive): thick peptidoglycan layer with embedded wall teichoic acids and lipoteichoic acids anchored in the plasma membrane; no outer membrane. Right panel (Gram-negative): thin peptidoglycan in the periplasmic space between inner membrane and outer membrane; outer membrane showing asymmetric bilayer with LPS (Lipid A, core polysaccharide, O-antigen), porins, and Braun's lipoprotein. All components clearly labeled with arrows.
### IV. Structures External to the Cell Wall
#### A. Capsules and Slime Layers (Glycocalyx)
A **capsule** is an organized, firmly attached layer surrounding the cell, usually composed of polysaccharide (though *Bacillus anthracis* produces a polypeptide capsule of poly-D-glutamic acid). Capsules are major virulence factors because they are **antiphagocytic**, shielding the bacterium from engulfment by immune cells, as exemplified by *Streptococcus pneumoniae*. Capsules also protect against desiccation and promote biofilm formation and adherence to surfaces. They can be detected by negative staining with India ink or by the capsule-swelling reaction known as Quellung. A **slime layer** is a more diffuse, loosely attached glycocalyx that facilitates adherence to surfaces, playing a role in dental plaque formation and in colonization of medical devices.
#### B. Flagella
Flagella are long, helical protein appendages that provide motility. They are composed of the protein **flagellin**, which also functions as the H antigen in serotyping. Structurally, each flagellum consists of a hollow helical **filament** of flagellin subunits, a curved **hook** that connects the filament to the **basal body**, and the basal body itself -- a motor complex embedded in the cell envelope. In Gram-negative bacteria the basal body comprises four rings (L, P, MS, and C) along with stator and rotor components, while in Gram-positive bacteria only two rings (MS and C) are present because there is no outer membrane to accommodate the L and P rings. Flagellar rotation is powered by the proton motive force or, in some species, by a sodium ion gradient. Counterclockwise rotation bundles flagella together and propels the cell forward in smooth "runs," while clockwise rotation causes the bundle to fly apart, producing "tumbles" that reorient the cell.
Flagellar arrangement varies among species. **Monotrichous** bacteria have a single flagellum at one pole, **amphitrichous** cells bear one flagellum at each pole, **lophotrichous** species have a tuft of flagella at one pole, and **peritrichous** bacteria are covered with flagella distributed over the entire surface. Spirochetes represent a special case, possessing **endoflagella (axial filaments)** that reside in the periplasmic space and generate the corkscrew motility characteristic of these organisms.
#### C. Pili (Fimbriae)
**Fimbriae** are short, hair-like protein appendages composed of pilin protein that mediate adherence to host cells and surfaces, making them important virulence factors. For example, the type 1 fimbriae of *E. coli* bind mannose residues on uroepithelial cells, contributing to urinary tract infections. The **sex pilus (F pilus)** is longer and fewer in number per cell. Encoded by the F (fertility) plasmid, it mediates conjugation -- the direct transfer of DNA between bacterial cells. **Type IV pili** are versatile structures involved in twitching motility, DNA uptake during natural competence, and adhesion to host tissues.
A detailed diagram of a Gram-negative bacterial flagellum. Panel A: Full-length view showing the filament (labeled as flagellin polymer), the hook, and the basal body embedded in the cell envelope (outer membrane, peptidoglycan, and inner membrane layers visible). Panel B: Enlarged cross-section of the basal body showing the L ring (in outer membrane), P ring (in peptidoglycan), MS ring (in inner membrane), C ring (cytoplasmic), Mot proteins (stator), and FliG/FliM/FliN switch complex. Panel C: Diagram of flagellar rotation -- CCW producing a bundle and forward run vs. CW producing tumbling, with directional arrows.
### V. Cytoplasmic Structures
#### A. Nucleoid
The nucleoid is the region of the cytoplasm that contains the bacterial chromosome. It is not bounded by a membrane. Typically, a bacterium has a single, circular, double-stranded DNA molecule that is haploid. This chromosome is supercoiled and compacted by nucleoid-associated proteins such as HU, IHF, H-NS, and Fis, while DNA topoisomerases regulate the degree of supercoiling.
#### B. Plasmids
Plasmids are small, circular, extrachromosomal DNA molecules that replicate independently of the chromosome. Although not essential for growth under normal conditions, plasmids often confer selective advantages. R plasmids carry antibiotic resistance genes, virulence plasmids encode toxins and adhesins, metabolic plasmids enable the degradation of unusual substrates, and the F plasmid mediates fertility and conjugation.
#### C. Ribosomes
Bacterial ribosomes are **70S** (in contrast to the 80S ribosomes of eukaryotes), composed of a **30S small subunit** (containing 16S rRNA and 21 proteins) and a **50S large subunit** (containing 23S rRNA, 5S rRNA, and 31 proteins). They are the site of protein synthesis and serve as targets for numerous antibiotics: aminoglycosides bind the 30S subunit, while macrolides and chloramphenicol target the 50S subunit.
#### D. Inclusion Bodies / Storage Granules
Bacteria accumulate a variety of inclusion bodies that serve as reserves of nutrients or fulfill specialized metabolic functions. **Poly-beta-hydroxybutyrate (PHB)** granules store carbon and energy. **Polyphosphate granules** (also called volutin or metachromatic granules) serve as phosphate and energy reserves and are characteristic of *Corynebacterium diphtheriae*. **Glycogen granules** provide another form of carbon and energy storage. **Sulfur globules** accumulate in sulfur-oxidizing bacteria. More specialized structures include **magnetosomes** -- membrane-bound magnetite crystals that enable magnetotaxis in organisms like *Magnetospirillum* -- **gas vesicles** that provide buoyancy in aquatic bacteria and archaea, and **carboxysomes**, polyhedral protein shells containing RuBisCO for carbon dioxide fixation.
### VI. Endospores
Endospores are extraordinarily resistant, dormant structures produced by certain Gram-positive genera, most notably *Bacillus*, *Clostridium*, and *Sporosarcina*. They form through the process of sporulation, triggered by nutrient depletion. From the outside in, an endospore may possess an **exosporium** (a thin, loose outermost covering not present in all species), a **spore coat** (multiple protein layers resistant to chemicals and enzymes), a **cortex** (a thick layer of modified peptidoglycan), a **core wall** (normal peptidoglycan), and a **core** (dehydrated cytoplasm containing DNA, ribosomes, dipicolinic acid chelated with calcium ions, and small acid-soluble spore proteins, or SASPs, that bind and protect DNA).
The remarkable resistance of endospores arises from several features: the dehydrated core reduces water activity, the DPA-calcium complex stabilizes DNA, SASPs protect DNA from UV radiation, heat, oxidation, and chemicals, and the thick spore coat is impervious to many chemical agents. As a result, endospores can survive boiling, radiation, desiccation, and chemical disinfection for years to centuries.
**Germination** occurs when conditions become favorable. Triggered by heat, specific nutrients, or mechanical damage, the endospore rehydrates and resumes vegetative growth. The clinical significance of endospore-forming bacteria is considerable: *Clostridium botulinum* causes botulism, *C. tetani* causes tetanus, *C. difficile* and *C. perfringens* cause gastrointestinal and soft tissue infections, and *Bacillus anthracis* is the causative agent of anthrax.
A cross-sectional diagram of a bacterial endospore within a vegetative cell. Panel A: The sporangium (mother cell) with a developing endospore inside, labeled to show exosporium, spore coat, cortex, core wall, inner membrane, and core (containing DNA bound by SASPs, ribosomes, DPA-Ca2+ complex). Panel B: A step-by-step schematic of the sporulation cycle (stages 0 through VII): vegetative cell, axial filament formation, asymmetric septation, engulfment, cortex synthesis, coat assembly, maturation, and lysis of mother cell to release the free spore. Panel C: Photomicrograph (or schematic) showing endospores stained green (malachite green) within pink vegetative cells (safranin counterstain), with arrows indicating terminal, subterminal, and central spore positions.