Microbiology & Molecular Biology Comprehensive Notes

Importance of Microbiology

• Diagnose, treat & prevent disease; outbreak preparedness; antimicrobial control.
• One-Health perspective: Humans ⟷ Animals ⟷ Ecosystem; Global & food security implications.
• Microbes can be:
  • Beneficial → bread, alcohol, vitamin synthesis, nitrogen fixation, biogas, bioremediation.
  • Harmful → disease, food spoilage.
• Ecosystem services:
  • Nitrogen cycle: $\text{N}<em>2 \xrightarrow{\text{Nitrogen fixers}} \text{NH}</em>3 \xrightarrow{\text{Nitrosomonas}} \text{NO}<em>2^- \xrightarrow{\text{Nitrobacter}} \text{NO}</em>3^-$
  • Natural-gas generation & pollutant degradation.

Microscopy Techniques

• Light compound microscope → staining required.
• Electron microscopy:
  • TEM (Transmission / tomography) → internal ultrastructure.
  • SEM (Scanning) → surface topology; specimen coated in inert metal (Au).

Taxonomy & Systematics

• All living cells share plasma membrane, ATP generation, genetic material; depend on 6 major elements (C, H, O, N, S, P).
• Historical schemes:
  • Linnaeus: 2 kingdoms (Animalia, Vegetabilia); binomial nomenclature (Genus species) based on morphology, geography, disease, etc.
  • Species concept → interbreeding fertile offspring.
• Modern phylogeny → $16S/18S$ rDNA sequencing (highly conserved) → 3 domains: Archaea, Bacteria, Eukarya (Woese).
• Identification toolbox:
  • Culture & staining → Gram, acid-fast.
  • Biochemical assays → ELISA.
  • Serology → agglutination.
  • Phage typing.
  • Molecular → PCR, DNA hybridization, MALDI-TOF mass spec.

Evolution & Spontaneous Generation Debate

• Abiogenesis hypothesis vs biogenesis (Pasteur’s swan-neck flask disproved SG).
• Protocells: self-organized amino-acid assemblies energized by sunlight.
• Pasteur milestones: pasteur effect (fermentation), germ theory, pasteurization, rabies vaccine (Joseph Meister), concept of attenuated vaccines (“serendipity”).
• Koch’s postulates (4 criteria) linking microbes to disease.

Historical Milestones & Pioneers

• Janssen → first compound microscope (3–9×).
• Hooke → micro-fungus (Mucor); Leeuwenhoek → protozoa, bacteria, RBC, sperm.
• Redi & Spallanzani disproved SG; Needham supported SG.
• Lister → antiseptic surgery (phenol); Jenner → cowpox vaccination; Lady Montagu → variolation.
• Virchow → veterinary pathology & zoonosis.
• Fleming → lysozyme; discovery of penicillin.
• Cohn → bacterial morphology groups; Chatton → prokaryote vs eukaryote.
• Metchnikoff → phagocytosis & innate immunity.
• Grassi → parasites; Bruce → Brucellosis & trypanosomes.
• Ivanowsky & Beijerinck → viruses (Tobacco Mosaic).
• Linnaeus (classification) & Woese (three domains).

Molecular Biology: Key Discoveries

• Griffith → transformation; Avery–MacLeod–McCarty → DNA = heredity.
• Watson & Crick → double helix; Rosalind Franklin → X-ray diffraction; Chargaff → $A=T,\; C=G$.
• Kary Mullis → PCR.

Central Dogma & Requirements for Genetic Material

• $DNA \xrightarrow{\text{transcription}} RNA \xrightarrow{\text{translation}} Protein$
• Properties: information storage, transmissibility, high-fidelity replication, capacity for variation.

Transcription (Prokaryotic Standard)

• Stages:
  • Initiation (RNA polymerase binds promoter).
  • Elongation → RNA built $5' \to 3'$ from template strand.
  • Termination at terminator sequence.
• Bacterial mRNA ≈ ready for translation immediately (no processing).
• Eukaryotic processing: intron splicing, 5' cap, 3' poly-A tail.

Translation Basics

• Genetic code read in 3-base codons.
• Start: AUG (Met); Stops: UAA, UAG, UGA.
• tRNA anticodon delivers specific amino acids to ribosome.

Nucleic Acid Structure

• DNA: double-stranded, antiparallel (template $3'\to5'$ / complementary $5'\to3'$).
  • Nucleotide = phosphate + deoxyribose + base (A, G = purines; C, T = pyrimidines).
  • Sugar-phosphate backbone via phosphodiester bonds.
• RNA: single-stranded; ribose; U replaces T.
• Carbon numbering: base on $1'$, phosphate on $5'$.
• RNA types: mRNA (message), rRNA (ribosome), tRNA (adapter; has anticodon).

Genome Components

• Genome = all genetic material, incl. mitochondrial DNA, plasmids, plastids.
• Mitochondria: ATP, FA & carbohydrate catabolism; endosymbiotic origin.
• Plasmid: small circular dsDNA, autonomous replication, horizontal transfer, AMR genes.
• Plastid: double-membrane plant/algal organelle; chemical storage; apicoplast in malaria (drug target).

DNA Replication

• Semi-conservative: parent strands template daughter strands.
• Origin(s) of replication:
  • Bacteria → single origin; Eukaryotes → multiple.
• Bidirectional fork movement $5'\to3'$.
  • Leading strand continuous; lagging → Okazaki fragments.
• Enzymes: helicase (unwind), topoisomerase (relieve supercoil), DNA polymerase, ligase.

Molecular Detection & Quantification

• Blotting: Southern (DNA), Northern (RNA), Western (protein).
• PCR reagents: template DNA, primers, dNTPs, Taq polymerase.
  • Thermal cycles: denaturation → annealing → extension (30–45 ×).
• Variants: qPCR (real-time), dPCR (absolute copy number), RT-PCR (RNA template).

Recombinant DNA & Cloning

• Combine DNA from different species → insert into host → expression/propagation.

Immunology Overview

• Immune system prevents, limits or eliminates pathogens by blocking entry, neutralizing invaders, or destroying altered self-cells.
• Immunity: resistance; susceptibility = vulnerability.

Innate Immunity (Non-Specific)

First Line – Barriers

• Physical: skin (epidermis/dermis), mucous membranes, shedding epithelium.
• Chemical: tears, saliva, gastric acid, mucus pH.
• Normal microbiota compete for nutrients (colonization resistance).

Second Line – Internal Defenses

• Phagocytes: neutrophils, eosinophils, monocytes/macrophages.
• Inflammation (redness, heat, pain, swelling, loss of function).
• Fever (elevated temp inhibits microbes).
• Antimicrobial proteins: complement cascade, interferons, iron-binding proteins.

Complement Pathways

• Classical, alternative, lectin.
• Outcomes:
  • MAC (C5–C9) → lysis.
  • Opsonization (C3b) → enhanced phagocytosis.
  • Chemotaxis & inflammation amplification.

Interferons

• Cytokines released by virus-infected cells; induce antiviral state in neighbors.
• Iron-binding proteins sequester Fe, limiting bacterial growth.

Phagocytosis Stages

1. Chemotaxis.
2. Adherence (TLR–PAMP binding).
3. Ingestion → phagosome.
4. Digestion → phagolysosome.
5. Elimination → residual body exocytosis.

Adaptive Immunity (Specific / Third Line)

• Triggered when pathogen escapes innate barriers; exhibits specificity & memory.
• Lymphoid & myeloid lineage from bone-marrow stem cells.

Lymphoid Organs

• Primary: bone marrow (B-cell maturation) & thymus (T-cell maturation).
• Secondary: lymph nodes, spleen, MALT – sites where mature lymphocytes encounter antigen.

Cellular Players

• B cells → antibody (humoral) response; plasma cells secrete Ig.
• T cells:
  • Helper (CD4, recognize MHC II) → cytokine orchestration.
  • Cytotoxic (CD8, recognize MHC I) → kill infected cells.
• NK cells → innate lymphoid cytotoxicity.

Antibody Classes

• IgG → highest concentration; placental transfer.
• IgM → first responder.
• IgE → allergy & parasitic response.

Humoral vs Cell-Mediated

• Humoral: Th cells activate B cells → plasma cells → antibodies.
• Cell-mediated: Th cells bind APC MHC II → cytokines → activate macrophages, NK, Tc cells → lysis of target.

Antigen Concepts

• Antigen = molecule provoking immune response; epitope = specific recognized part.
• Hapten: small molecule immunogenic only when attached to carrier (e.g., penicillin allergy).
• Secondary (anamnestic) response = faster, stronger due to memory cells.

Eukaryotic vs Prokaryotic Transcription

• Core mechanics conserved; key differences:
  • 3 RNA polymerases (I: rRNA; II: mRNA; III: tRNA & others) in eukaryotes.
  • RNA Pol II requires 5 general transcription factors.
  • Extensive RNA processing occurs only in eukaryotes.