Bacterial Cell Wall, Peptidoglycan & Gram-Positive/Negative Structures – Comprehensive Lecture Notes

Course & Assessment Logistics

• Blackboard holds all posted materials; recordings contain in-class quiz questions.

• Graduate‐level expectation: no external study guides; responsibility on students to master content.

• First quiz (Biochem) scheduled Monday; Micro quiz questions mirror in-class practice (MCQs, vignette style).

• Class rescheduled to Thursday (01/23) this week; check Blackboard calendar.

Recap of Bacterial Cell Anatomy (Day 1)

• Cell envelope (excluding wall):

• Cytoplasmic (plasma) membrane

• External layers: capsule, pili (fimbriae), flagella
  • Cytoplasm: aqueous matrix containing inclusion bodies, ribosomes, nucleoid.
  • Nucleoid:

• Single, circular, double-stranded, haploid chromosome.

• Extra-chromosomal DNA (plasmids) provide antibiotic resistance & stress survival.
  • Ribosomes (70 S):

• Sedimentation coefficients: 50 S (23 S + 5 S rRNA) + 30 S (16 S rRNA).

• 23 S & 16 S rRNAs used for genotyping; major antibiotic targets (e.g., tetracycline).

• Term S = Svedberg unit (sedimentation rate, not molecular mass).
  • Inclusion bodies: storage granules (glycogen, starch, poly-β-hydroxybutyrate, polyphosphate/“volutin”); useful in identification (e.g., Corynebacterium diphtheriae metachromatic granules).

Bacterial Spores (Endospores)

• Produced mainly by Gram-positive genera (Bacillus, Clostridium).
• Trigger: nutrient depletion during transition from log → stationary phase; proteases destroy sporulation repressor → sporulation genes expressed.
• Dormant/vegetative distinction

• Vegetative = metabolically active & dividing.

• Dormant (spore) = metabolically inert, non-dividing seed.
  • Clinical relevance: spores resist heat, drying, chemicals; can persist through therapy ⇒ relapse years later.

Spore Architecture

Sporulation Steps (microscopy analogy)

1. Axial filament forms after chromosome replication.

2. Septum near pole ⇒ forespore.

3. Engulfment by mother cell (double membrane).

4. Cortex & coat laid; Ca-dipicolinate incorporated.

5. Mother cell lyses → mature spore released.

Germination

1. Activation (heat, abrasion, age).

2. Germination agents (water, nutrients e.g., alanine) break cortex.

3. Outgrowth → vegetative cell division.

Bacterial Growth Curve & Treatment Timing

• Lag → Log → Stationary → Death phases.

• Spore initiation begins late log/early stationary.

• Optimal antibiotic window: log phase (maximal vegetative growth).

In-Class Practice MCQs (Key Takeaways)

• Serotype = subgroup distinguished by surface antigens detected by antisera (NOT gram stain, shape, or genotype alone).

• Endospore resistance due to thick coat + high Ca-dipicolinic acid in core.

• Capsule functions: immune evasion (anti-phagocytic), adherence—not ATP generation.

• Pili (fimbriae): attachment; F-pili: conjugative DNA transfer.

• 70 S ribosome unique to prokaryotes → antibiotic target.

• Gram-negative rods stain pink; rods = bacilli.

• Gram-negative appear pink because thin PG loses crystal violet during decolorization, then safranin counter-stains.

• Primary Gram stain = crystal violet.

Cell Wall: Overview & Significance

• Provides shape, rigidity, osmotic protection; unique to bacteria → drug target.

• Main constituent = peptidoglycan (PG, murein, mucopeptide).

• Only exception: Mycoplasma (no PG; sterol-rich membrane).

Peptidoglycan Monomer

• Disaccharide-pentapeptide unit:
\text{N-acetylglucosamine (NAG)}\;–\;\beta1\rightarrow4\;–\;\text{N-acetylmuramic acid (NAM)}–\text{(L-Ala–D-Glu–X–D-Ala–D-Ala)}
• Position 3 X = \text{L-Lys} (Gram +) or \text{meso-DAP} (Gram –).
• Peptide not synthesized by ribosomes—assembled enzymatically.

Cross-linking Differences

• Gram +: L-Lys (3) ↔ D-Ala (4) via pentaglycine bridge; ⇒ ~40 PG layers.

• Gram –: meso-DAP (3) directly ⇄ D-Ala (4); ⇒ 1–2 PG layers.

PG Biosynthesis (three compartments)

1. Cytoplasm

   • UDP-NAG → UDP-NAM; sequential addition of pentapeptide.

2. Membrane (inner)

   • Carrier bactoprenol-P binds NAM-pentapeptide ((\uparrow!P_i) high-energy pyrophosphate liaison).

   • NAG added → complete monomer; Flippase (FlipA) translocates complex to outer leaflet.

   • Bactoprenol must be de-phosphorylated to recycle; bacitracin blocks this step (binds bactoprenol-PP).

3. Periplasm/exterior

   • Transglycosylase forms \beta1\rightarrow4 glycosidic link between new monomer & glycan chain.

   • Transpeptidase (PBP) cross-links peptides (D-Ala lost) ⇒ rigid mesh.

Antibiotic Targets

• \beta-lactams (penicillin, cephalosporin): inhibit PBPs ⇒ block transpeptidation.

• Glycopeptides (vancomycin): bind D-Ala-D-Ala termini, hinder PBPs (Gram + only).

• Bacitracin: prevents bactoprenol de-phosphorylation.

• Lysozyme (host enzyme): hydrolyses \beta1\rightarrow4 linkage; excess causes bacterial lysis.

Gram-Positive Wall Extras

• 40+ PG layers; teichoic acids (poly-ribitol/glycerol-phosphate) covalently linked to PG.

• Lipoteichoic acids anchored in cytoplasmic membrane—adhesion & PAMP trigger.

Gram-Negative Envelope

• Layers (inside → out):

1. Cytoplasmic membrane.

2. Thin PG (1–2 layers) within periplasmic space.

3. Outer membrane (OM)—asymmetric:

   • Inner leaflet: phospholipid + Braun lipoprotein (anchors PG).

   • Outer leaflet: lipopolysaccharide (LPS).

Periplasmic Space

• Contains hydrolytic enzymes, binding proteins, antibiotic-inactivating enzymes, virulence factors (hyaluronidase).

Porins (OMP C/F etc.)

• Trimeric β-barrel channels; permit diffusion of small hydrophilic solutes; restrict large or hydrophobic drugs.

Lipopolysaccharide (LPS) – Endotoxin

1. Lipid A: phosphorylated GlcN disaccharide + \beta-hydroxymyristic FAs; responsible for toxicity.

2. Core polysaccharide: inner + outer core (KDO, heptoses).

3. O-antigen: repeating sugar units (≈40); highly variable → serotyping, immune evasion.

Pathophysiology

• Constantly shed; binds LPS-BP → TLR-4 on macrophages/B-cells.

• Cytokine storm: \text{IL-1},\;\text{TNF-\alpha},\;\text{IL-6} etc.

• Low dose: fever, localized inflammation.

• High dose (bacteremia):
  • Complement & coagulation cascades ⇒ disseminated intravascular coagulation (DIC).
  • Vasodilation, capillary leak ⇒ hypotension, multi-organ failure (Schwartzman reaction).
  • Clinical term: septic shock.

LOS (Lipo-oligosaccharide)

• Similar to LPS but lacks O-antigen; found in Neisseria, Haemophilus.

Special Cell-Wall Variants

• Mycobacterium: PG linked to arabinogalactan/mycolic acids → acid-fast wall.

• Mycoplasma/Ureaplasma: no PG; contain sterols; insensitive to β-lactams.

Metaphors & Illustrative Analogies

• Lego® bricks: NAG-NAM-pentapeptide monomers snapped into existing wall.

• Pregnant cell: mother bacterium engulfs forespore, then sacrifices itself.

• Seeds vs plants: spore (seed) germinates in moist, nutrient-rich soil (host).

Connections & Real-World Relevance

• Antibiotics exploit structural differences (70 S vs 80 S ribosome; PG synthesis enzymes; unique LPS) to achieve selective toxicity.

• Spore persistence explains latent/recurrent Clostridioides difficile or anthrax cases.

• LPS-induced sepsis major cause of ICU mortality; underscores need for prompt Gram-negative coverage.

Ethical & Practical Implications Discussed

• Overreliance on instructor-made study guides counterproductive in graduate training; cultivate self-directed learning.

• Misuse of antibiotics accelerates resistance; understanding mechanisms guides rational therapy.

• Infection control: spore-forming organisms withstand routine disinfection—necessitate sporicidal agents.

Quick-Reference Numbers & Equations

• Ribosomes: 70\,S = 50\,S + 30\,S (prokaryote); 80\,S = 60\,S + 40\,S (eukaryote).
• PG glycosidic bond: \text{NAG} \;\beta1\rightarrow4\; \text{NAM}.
• Bacterial doubling time (example): E. coli ≈ 30\;\text{min}.

Exam/Quiz Study Tips (from Q&A)

• Review lecture recordings for embedded MCQs.

• Formulate your own notes; use textbook (Ryan & Sherris microbiology) ch 11 (v10) / ch 12 (v9).

• Supplement with practice banks (Princeton, etc.).