Bacterial Classification, Structure, and Replication

Overview and Size Comparisons

• Bacteria represent the smallest cellular organisms and are visible primarily with light or electron microscopy.

• Size spectrum of bacteria:

  • Smallest bacteria (Chlamydia and Rickettsia): $0.1$ to $0.2\,\mu\text{m}$ in diameter.

  • Average bacterial cell: Approximately $1\,\mu\text{m}$ in diameter (resolvable using a light microscope, which has a resolution limit of $0.2\,\mu\text{m}$).

  • Giant bacteria: Newly described species can be hundreds of times larger than standard bacteria and are visible to the naked eye.

• Size comparisons with eukaryotes:

  • Red blood cells: $7\,\mu\text{m}$ in diameter.

  • Nerve cells: Range up to several feet in length.

Differences Between Eukaryotes and Prokaryotes

• Taxonomic Categorization:

  • Eukaryotes ("true nucleus"): Animals, plants, fungi, algae, and protozoa.

  • Prokaryotes ("primitive nucleus"): Bacteria, Archaea, and blue-green algae.

  • Archaea (Archaebacteria): Resemble bacteria structurally in most aspects but represent a distinct domain evolutionary separate from bacteria and eukaryotes.

• Nuclear and Genomic Differences:

  • Eukaryotes: Enclosed classic nuclear membrane, diploid genome consisting of multiple linear DNA strands. Humans possess $2$ copies of $23$ chromosomes ($2.9 \times 10^9$ base pairs, measuring $990\,\text{mm}$ in total length).

  • Prokaryotes: No nuclear membrane. Haploid genome consisting of a single, double-stranded, supercoiled circular DNA chromosome located in a nucleoid region. Typical bacterium (Escherichia coli) chromosome contains approximately $5\,\text{million}$ base pairs ($5000\,\text{kilobase [kb]}$ pairs) with a length of $1.3\,\text{mm}$ (nearly $1000$ times the diameter of the cell). Mycoplasma chromosomes represent the smallest bacterial genomes, approximately $\frac{1}{4}$ the size of E. coli.

• Cytoplasmic Structures and Organelles:

  • Eukaryotes: Contain membrane-bound organelles including mitochondria (site of cellular respiration), Golgi apparatus, endoplasmic reticulum (smooth and rough), and lysosomes. Contain $80\text{S}$ ribosomes (subunits $60\text{S} + 40\text{S}$).

  • Prokaryotes: Lack membrane-bound organelles (mitochondria, Golgi bodies, endoplasmic reticulum). Respiration occurs via the cytoplasmic membrane. Contain $70\text{S}$ ribosomes (subunits $50\text{S} + 30\text{S}$), which differ significantly in RNA and protein content from eukaryotic ribosomes, forming primary targets for antibacterial therapies.

• Membrane and Cell Envelope Features:

  • Cytoplasmic Membrane: Eukaryotic membranes contain sterols (e.g., cholesterol). Prokaryotic membranes do not contain sterols, with Mycoplasma being the sole exception.

  • Cell Wall: Eukaryotic cell walls are absent in animals/protozoa and present in fungi. Prokaryotic cell wall is a complex structure composed of peptidoglycans, proteins, and lipids.

  • Reproduction: Eukaryotes reproduce sexually or asexually. Prokaryotes reproduce strictly asexually via binary fission.

  • Motility: Eukaryotes utilize complex flagella if present. Prokaryotes utilize simple flagella driven by membrane potential.

Bacterial Classification

• Bacteria are classified using macroscopic appearance, microscopic appearance, metabolic/growth characteristics, antigenicity, and genetic analysis.

• Macroscopic and Microscopic Distinction:

  • Macroscopic Features: Growth on selective/nutrient media forms colonies (communities containing $\ge 10^6$ organisms). Colony traits include color, size, shape, smell, antibiotic resistance, sugar fermentation (e.g., lactose fermentation distinguishing E. coli from Salmonella), hemolytic properties (erythrocyte lysis), or lipid hydrolysis (e.g., clostridial lipase).

  • Microscopic Morphology:

  • Coccus: Spherical shape (e.g., Staphylococcus).

  • Bacillus: Rod shape (e.g., E. coli).

  • Spirillum / Spirochete: Snake-like or spiral shape (e.g., Treponema).

  • Filamentous: Branched morphology resembling fungi (e.g., Nocardia, Actinomyces).

  • Aggregates: Grapelike clusters (Staphylococcus aureus) or diplococci (pairs, e.g., Streptococcus, Neisseria).

• Gram Stain Procedure and Mechanism:

  • Complete procedure completed in under $10\,\text{minutes}$:

  1. Heat-fix or dry bacteria onto a glass slide.

  2. Stain with crystal violet primary stain.

  3. Precipitate stain by adding Gram iodine mordant.

  4. Wash with an acetone-based decolorizer and water.

  5. Apply safranin red counterstain.

  • Gram-Positive Bacteria: Retain crystal violet-iodine complex, appearing purple ("P-PURPLE-POSITIVE"). Caused by entrapment within a thick, cross-linked, meshlike peptidoglycan layer.

  • Gram-Negative Bacteria: Thin peptidoglycan layer cannot retain crystal violet following decolorizer treatment; cells accept safranin red counterstain, appearing red.

  • Limitations: Unreliable for starved/stationary-phase cultures or antibiotic-treated cells due to peptidoglycan degradation. Ineffective for Mycobacteria (waxy lipid outer shell requiring acid-fast stain) and Mycoplasma (lack peptidoglycan entirely).

• Metabolic, Antigenic, and Genetic Distinctions:

  • Metabolic Signature: Oxygen reliance (aerobic vs. anaerobic), specific nutrient demands, production of characteristic metabolic byproducts (acids, alcohols), or enzymes (e.g., staphylococcal catalase). Automated systems yield numerical biotypes.

  • Serotyping: Identification of specific surface antigens using antibodies. Crucial for organisms difficult (Treponema pallidum) or dangerous (Francisella) to culture, specific disease-associated strains (E. coli O157:H7), or rapid identification (Streptococcus pyogenes).

  • Genotypic Methods: Analysis of nucleic acid sequences using DNA hybridization, PCR amplification, plasmid analysis, ribotyping, and chromosomal DNA fragment analysis. Ribosomal DNA (rDNA) sequencing uses highly conserved sequences for genus/family level identification and variable sequences for species/subspecies identification.

Bacterial Structure

• Cytoplasmic Components:

  • Nucleoid: Contains single double-stranded circular DNA chromosome. Devoid of nuclear membrane, histones, or nucleosomes.

  • Coupled Transcription-Translation: Absence of nuclear membrane allows ribosomes to attach to mRNA and initiate protein synthesis while mRNA is actively being transcribed from DNA.

  • Plasmids: Small, circular extrachromosomal DNA molecules conferring selective advantages, such as antibiotic resistance genes.

  • Ribosomes: $70\text{S}$ total ($30\text{S}$ and $50\text{S}$ subunits).

  • Cytoplasmic Membrane: Phospholipid bilayer lacking sterols (except Mycoplasma). Functions in energy production/electron transport (equivalent to mitochondria), active metabolite transport, maintenance of membrane potential via ion pumps, and structural lining of inner membrane with actin-like protein filaments that define cell morphology and septum location.

• Gram-Positive Cell Wall:

  • Peptidoglycan Layer: Thick ($150$ to $500\,\text{\AA}$), multilayered, porous exoskeleton.

  • Lysozyme Susceptibility: Lysozyme (found in human tears and mucus) cleaves the glycan backbone. Cleavage in iso-osmotic media produces an osmotically fragile protoplast, which lyses in hypotonic conditions.

  • Teichoic and Lipoteichoic Acids: Water-soluble anionic polymers of polyol phosphates (ribitol phosphate or glycerol phosphate). Covalently bound to peptidoglycan (teichoic acid) or anchored in cytoplasmic membrane via a fatty acid (lipoteichoic acid). Functions include structural strengthening, calcium ion sequestration, serotype antigenicity, adherence, and activation of host innate immune responses.

  • Associated Proteins: M protein (Streptococcus), R protein (Staphylococcus), and C polysaccharides.

• Gram-Negative Cell Wall:

  • Peptidoglycan Layer: Thin layer representing only $5\%$ to $10\%$ of cell wall mass. Contains no teichoic or lipoteichoic acids.

  • Periplasmic Space: Compartment between cytoplasmic membrane and outer membrane containing nutrient transport proteins, degradative hydrolytic enzymes (proteases, phosphatases, lipases, nucleases), and pathogenic lytic factors (collagenases, hyaluronidases, $\beta$-lactamase).

  • Secretion Systems: Types I, II, III, IV, and V transport devices. Type III secretion system spans inner and outer membranes acting as a molecular syringe to inject virulence proteins directly into host cells.

  • Outer Membrane: Asymmetric lipid bilayer. Inner leaflet contains normal phospholipids; outer leaflet consists primarily of Lipopolysaccharide (LPS). Contains transmembrane porin proteins forming channels that allow passive diffusion of hydrophilic molecules smaller than $700\,\text{Da}$.

  • Stabilization and Disruption: Membrane integrity maintained by divalent cation ($\text{Mg}^{2+}$, $\text{Ca}^{2+}$) linkages between LPS phosphate groups. Chelation using EDTA/tetracycline or treatment with polymyxin disrupts the outer membrane. Addition of lysozyme to outer membrane-disrupted cells produces spheroplasts.

  • Lipopolysaccharide (LPS / Endotoxin) Structure:

  • Lipid A: Phosphorylated glucosamine disaccharide backbone with attached fatty acids. Anchors LPS in outer membrane. Responsible for endotoxic toxicity. Stimulates B cells and induces release of $\text{IL-1}$, $\text{IL-6}$, and $\text{TNF}$ from macrophages/dendritic cells, causing fever, shock, and the Shwartzman reaction (disseminated intravascular coagulation).

  • Core Polysaccharide: $9$ to $12$ sugars containing 2-keto-3-deoxy-octanoate (KDO) and phosphate groups.

  • O Antigen: Long linear polysaccharide chain of $50$ to $100$ repeating units ($4$ to $7$ sugars/unit). Extends outward; used for strain serotyping.

  • Lipooligosaccharide (LOS): Found in Neisseria species; lacks O antigen, making organisms more susceptible to complement-mediated killing.

• External Structures:

  • Capsule and Slime Layer (Glycocalyx): Polysaccharide layers (except Bacillus anthracis, which possesses a poly-D-glutamic acid polypeptide capsule). Loosely adherent layers are termed slime layers. Capsules act as major virulence factors by inhibiting phagocytosis and antibody/complement binding. Promotes tissue adherence (e.g., Streptococcus mutans dextran/levan synthesis for enamel adhesion).

  • Biofilms: Polysaccharide meshwork secreted by bacterial communities (Pseudomonas aeruginosa, S. aureus, S. mutans) triggered by quorum sensing. Protects bacteria against host immune defenses and antibiotics.

  • Flagella: Helically coiled protein propellers ($15$ to $20\,\text{nm}$ diameter) made of flagellin subunits. Driven by a membrane potential-powered ATP motor anchored via hook and basal body structures. Facilitates chemotaxis (straight swimming interspersed with tumbling). Acts as a ligand for Toll-like receptor 5 (TLR5).

  • Fimbriae / Pili: Non-coiled protein filaments ($3$ to $8\,\text{nm}$ diameter, up to $15$ to $20\,\mu\text{m}$ length) composed of pilin subunits. Uniformly distributed (peritrichous). Function as adhesins/lectins (binding specific sugars such as mannose). F pili (sex pili) encoded by F plasmid construct hollow tube for bacterial DNA conjugation.

Structure and Biosynthesis of Cell Wall Components

• Peptidoglycan Structure:

  • Alternating disaccharide backbone of N-acetylglucosamine (GlcNAc / NAG / G) and N-acetylmuramic acid (MurNAc / NAM / M) connected by $\beta-1,4$ glycosidic bonds.

  • Tetrapeptide attached to MurNAc containing alternating D- and L-amino acids.

  • Position $3$ diamino amino acid (lysine, diaminopimelic acid [DAP], or diaminobutyric acid) provides free amine group required for cross-linking.

  • Cross-linking occurs between the free amine at position $3$ and the D-alanine at position $4$ of an adjacent chain, releasing the terminal (5th) D-alanine. Gram-positive S. aureus utilizes a pentaglycine ($\text{gly}_5$) cross-bridge.

• Peptidoglycan Synthesis Phases:

  1. Phase 1 (Intracellular): Glucosamine converted to MurNAc, activated with UTP to form UDP-MurNAc. Sequential addition of amino acids constructs UDP-MurNAc-pentapeptide (ending in D-Ala-D-Ala).

  2. Phase 2 (Membrane): UDP-MurNAc-pentapeptide bound to hydrophobic membrane carrier bactoprenol (undecaprenol, a $\text{C}_{55}$ isoprenoid) via pyrophosphate linkage, releasing UMP. GlcNAc is attached. Modifications (e.g., pentaglycine bridge) occur here.

  3. Phase 3 (Translocation): Bactoprenol translocates the disaccharide-peptide monomer across cytoplasmic membrane to exterior surface.

  4. Phase 4 (Assembly and Cross-linking):

  • Transglycosylases insert disaccharide unit into peptidoglycan chain using energy from pyrophosphate bond cleavage.

  • Pyrophosphobactoprenol converted back to phosphobactoprenol for recycling; step specifically blocked by bacitracin.

  • Transpeptidation: Membrane-bound Penicillin-Binding Proteins (PBPs: transpeptidases and carboxypeptidases) catalyze peptide bond exchange between position $3$ amine (or pentaglycine) and position $4$ D-Ala, driving off the $5\text{th}$ D-Ala without requiring external ATP.

  • Antimicrobial mechanisms: $\beta$-lactams mimic D-Ala-D-Ala transition state structure to inhibit PBPs. Vancomycin directly binds D-Ala-D-Ala terminal residues to sterically block transpeptidation.

  • Autolysins (e.g., lysozyme-like enzymes) continuously cleave peptidoglycan to allow structural expansion during growth.

Bacterial Cell Division

• Chromosome replication triggers cell division.

• Membrane filament ring establishes midcell site for division septum formation.

• Septum consists of two cytoplasmic membranes separated by two layers of peptidoglycan.

• Morphological growth angles:

  • Linear chains (Streptococcus): Growth zones oriented at $180^\circ$.

  • Cluster formation (Staphylococcus): Growth zones oriented at $90^\circ$.

• Incomplete septum cleavage results in persistent cellular chains or clusters.

Endospores and Sporogenesis

• Spore-Forming Genera: Strictly limited to certain Gram-positive bacteria, including soil organisms Bacillus (Bacillus anthracis) and Clostridium (Clostridium tetani, Clostridium botulinum). Gram-negative bacteria never form spores.

• Spore Characteristics: Dehydrated, non-reproductive structure protecting genome in suspended animation against extreme heat, desiccation, radiation, pH extremes, enzymes, and chemical disinfectants. Can survive in environment for centuries.

• Spore Architecture:

  • Core: Contains complete chromosome copy, minimal essential proteins/ribosomes, and high concentration of calcium bound to dipicolinic acid.

  • Inner membrane.

  • Cortex: Inner thin layer of tightly cross-linked peptidoglycan surrounded by loose outer peptidoglycan layer.

  • Keratin-like protein coat and outer exosporium layer.

• Sporogenesis (Endospore Formation):

  • Triggered by nutrient depletion (e.g., loss of alanine) taking $6$ to $8\,\text{hours}$.

  • Involves gene transcription cascade, dipicolinic acid accumulation, chromosome duplication, septation, and deposition of multi-layered cortex and protein coat.

• Germination:

  • Activation triggered by mechanical disruption of spore coat, heat, pH, water, and triggering nutrients (e.g., alanine).

  • Duration: Approximately $90\,\text{minutes}$.

  • Spore swells, sheds protective coats, takes up water, and regenerates a single vegetative bacterium.

Questions & Clinical Applications

• Question 1: How does each of the differences between prokaryotes and eukaryotes influence bacterial infection and treatment?

  • Answer:

  • Size: Smaller size allows prokaryotes to colonize confined anatomical spaces. Smaller size necessitates a smaller genome.

  • Nuclear Structures: Lack of nuclear membrane couples replication, transcription, and translation. Inhibiting one process rapidly impacts others.

  • Chromosomes: Single circular haploid chromosome requires topoisomerases to relieve supercoiling stress, making these enzymes ideal targets for antibacterial agents (e.g., quinolones). Single-copy genes mean mutations directly alter phenotype due to lack of a diploid backup gene.

  • Ribosomes: The distinct $70\text{S}$ ($50\text{S} + 30\text{S}$) structure provides a selective drug target distinct from eukaryotic $80\text{S}$ ribosomes.

  • Cytoplasmic Membrane: Unique phospholipid composition makes membrane vulnerable to polymyxins. Maintenance of membrane potential drives essential ATP synthesis.

  • Cell Wall: Unique peptidoglycan matrix prevents osmotic lysis in fluids like water. Synthesis pathways provide targets for $\beta$-lactams, vancomycin, and bacitracin. Pili structures enable targeted tissue adherence (e.g., bladder epithelium).

• Question 2: How do the differences between gram-positive and gram-negative cell walls influence the cells' clinical behavior, detection, and treatment?

  • Answer:

  • Detection: Thick peptidoglycan traps crystal violet-iodine complexes in Gram-positive walls. Single-layer Gram-negative peptidoglycan loses stain during alcohol wash and requires safranin counterstain.

  • Clinical Behavior: Gram-negative outer membrane LPS (endotoxin) acts as a powerful activator of host innate immunity, driving high fever and septic shock.

  • Treatment: The Gram-negative outer membrane functions as a permeability barrier against complement attack, hydrophobic compounds, large molecules, and select antibiotics, preventing access to internal peptidoglycan targets.

• Question 3: List the cell wall components that contribute to virulence by protecting the bacteria from immune responses. List those that contribute to virulence by eliciting toxic responses in the human host.

  • Answer:

  • Immune Protection Factors: Peptidoglycan and LPS O-antigens restrict complement membrane attack complex access. Capsules block phagocytosis, antibody binding, and complement access. Surface proteins inhibit host defenses (e.g., Staphylococcus protein A binds IgG Fc regions; Streptococcus M protein inhibits phagocytosis).

  • Toxic Host Response Factors: LPS (endotoxin Lipid A) strongly activates Toll-like receptors and cytokine pathways. Teichoic acid and peptidoglycan serve as weaker activators of host Toll-like receptors.

• Question 4: When peptidoglycan synthesis is inhibited, what processes kill the bacteria? List the precursors that would build up within the bacteria if recycling of bactoprenol were inhibited by penicillin, vancomycin, or bacitracin.

  • Answer:

  • Cell Death Mechanism: Autolysins continuously degrade existing peptidoglycan to allow cell growth. Inhibiting new synthesis weakens the cell wall relative to internal osmotic pressure, leading to cell lysis.

  • Accumulating Precursors: Inhibition causes cytoplasmic accumulation of UDP-MurNAc-pentapeptide (NAG-NAM-pentapeptide containing terminal D-Ala-D-Ala) due to blocked transpeptidation or lack of available bactoprenol carriers.

• Question 5: Why are spores more resistant to environmental stresses?

  • Answer: Spores are metabolically dormant, dehydrated, packed with calcium-dipicolinic acid complexes, and encased within multiple protective peptidoglycan cortex layers and tough keratin-like protein coats.

• Question 6: The laboratory would like to selectively eliminate gram-positive bacteria from a mixture of gram-positive and gram-negative bacteria. Which of the following procedures would be more appropriate and why or why not?

  • a. Treatment with ethylenediaminetetraacetic acid (a divalent cation chelator)

  • b. Treatment with mild detergent

  • c. Treatment with lysozyme

  • d. Treatment with transpeptidase

  • e. Treatment with ampicillin (a hydrophilic $\beta$-lactam antibiotic)

  • Answer:

  • Correct Choice: c (Treatment with lysozyme).

  • Rationale:

    • EDTA (a) disrupts Gram-negative outer membranes with minimal impact on Gram-positive walls.

    • Mild detergent (b) harms Gram-positive cells more, but lacks absolute selectivity.

    • Lysozyme (c) directly degrades exposed Gram-positive peptidoglycan causing rapid lysis, whereas the Gram-negative outer membrane acts as a physical barrier preventing lysozyme access to peptidoglycan.

    • Transpeptidase (d) is an enzyme, not an inhibitor; it causes no cell death.

    • Ampicillin (e) passes through Gram-negative porins, killing both Gram-positive and Gram-negative organisms indiscriminately.