Definitive Study Guide: Introductory Microbiology and Molecular Biology

Foundations of Microbiology and Early Life

• Categories of Microorganisms: Studied by microbiologist to understand basic life processes.
• Rationale for Study: Microbes are studied because they are simple, reproduce quickly, and grow densely.
• Microbial Dimensions:
      * Cellular Microbes: Generally smaller than $1\,\text{millimeter}$ and often unicellular.
      * Multicellular Microbes: Distinguished by a lack of differentiated tissues.
• Characteristics of Life: Must possess the ability to evolve, obtain and use energy (e.g., metabolism), and reproduce.
• Chronology of Earth and Life:
      * Age of Earth: Approximately $4.5\,\text{to}\,4.6\,\text{billion years old}$.
      * Origin of Life: Roughly $3.5\,\text{billion years ago}$.
      * Evidence: Indirect evidence found in molecular fossils, which are chemicals in rock or sediment related to molecules in modern cells.
• Stromatolites:
      * Definition: Layered, often dome-shaped rocks.
      * Formation: Created by the incorporation of mineral sediments into layers of microorganisms growing as thick mats on surfaces.
      * Age: Date back to approximately $3.5\,\text{BYA}$.
      * Location: Primarily found in shallow water.
      * Significance: Represents the earliest fossil evidence of life on Earth.

The Origin of Life and the RNA World

• Early Earth Conditions: Characterized by an absence of oxygen; biotic molecules were initially created abiotically.
• Microbial Dominance: For the first $3/4$ of life's history, the only living organisms were microbial.
• RNA World Hypothesis:
      * Origin: Term coined by Gilbert.
      * Description: Refers to a precellular stage in evolution.
      * Capabilities of RNA: It can store information (like DNA) and act as a catalyst (like proteins/enzymes), serving both functions simultaneously.
• LUCA (Last Universal Common Ancestor):
      * Located on the bacterial branch of the tree of life.
      * All life forms arose from a single common ancestor.
      * Archaea and Eukarya evolved separately from the bacterial lineage.

Historical Figures in Microbiology

• Robert Hooke: The first to describe molds.
• Antoni van Leeuwenhoek: The first to describe bacteria.
• Louis Pasteur: Famous for disproving the theory of spontaneous generation.
• Robert Koch: Demonstrated that bacteria cause disease; developed techniques for obtaining pure cultures. He famously drank $H.\,pylori$ to prove it causes stomach disease.
• Sergei Winogradsky: Discovered chemoautotrophy (obtaining energy from chemicals) and was the first to study microbes within their environmental context.

Key Experimental Protocols and Postulates

• Pasteur's Experiment: Tested if sterile nutrient broth could spontaneously generate life.
      1. Nutrient broth was added to flasks with S-shaped necks and boiled to kill existing microbes.
      2. In Experiment #1, the neck was broken, exposing broth to air.
      3. In Experiment #2 (control), the neck remained intact; dust particles trapped in the curve could not reach the broth.
      4. Result: Broth in the broken neck became cloudy (microbial growth), while the unbroken neck remained clear. This disproved spontaneous generation.
• Koch's Postulates: Four criteria to identify the causative agent of a disease:
      1. The pathogen must be present in all cases of the disease.
      2. The pathogen must be isolated from the host and grown in a pure culture.
      3. The pathogen from the pure culture must cause disease when inoculated into a healthy, susceptible lab animal.
      4. The pathogen must be re-isolated from the new host and shown to be identical to the original pathogen.
• Drake Equation: A mathematical framework used to estimate the number of communicating civilizations in space based on specific parameters.

Microscopy and Staining Techniques

• Resolution: The ability of a lens to separate or distinguish between two small objects that are close together.
• Gram Staining Procedure:
      * Step 1: Stain with crystal violet for $1\,\text{minute}$, rinse with water. All cells appear purple.
      * Step 2: Add iodine for $1\,\text{minute}$, rinse. Cells remain purple.
      * Step 3: Decolorize with alcohol for $10\text{--}30\,\text{seconds}$, rinse. Gram (+) remain purple; Gram (-) become colorless.
      * Step 4: Counterstain with safranin for $1\,\text{minute}$, rinse, and blot dry. Gram (+) are purple; Gram (-) are red/pink.
• Microscopy Types:
      * Bright-field: Image relies on density differences between specimen and surroundings.
      * Phase-contrast: Improves contrast without staining; shows dark cells on a light background. Advantage: Cells remain alive.
      * Dark-field: Light is shone from the side and scattered. Excellent for observing motility. Features a dark background and light object.
      * Fluorescence: Uses emitted light to create images. DAPI is a generic stain that binds to all DNA and fluoresces blue, used when specimens do not autofluorescence.
      * Atomic Force Microscopy: Creates 3D maps by dragging a stylus over the specimen to measure weak repulsive forces. Can visualize contours of the cell membrane.
      * Confocal Scanning Laser Microscopy (CSLM): Uses lasers to image layers that stack into a 3D image. Resolution is approximately $0.1\,\text{micrometer}$.
      * Electron Microscopy (EM): Uses electron beams and electromagnetic lenses in a vacuum. High magnification ($15\text{X to }100,000\text{X}$) and resolution.
          * TEM (Transmission): Specimen is very thin and metal-stained; cells are dead.
          * SEM (Scanning): Specimen coated in heavy metal; beam scans the object surface.
• FISH (Fluorescence In Situ Hybridization):
      1. Create a fluorescent or modified copy of a DNA probe sequence.
      2. Denature target and probe sequences with heat or chemicals to allow new H-bond formation.
      3. Mix probe and target; the probe hybridizes to its complementary sequence on the chromosome.
      4. Visualize the hybrid using a fluorescence microscope to identify and count similar DNA in a sample.

Molecular Components of the Cell

• Lipids: Amphipathic molecules (polar and non-polar) used for cell membranes and energy storage.
• Carbohydrates: Contain C, H, and O in a $1:2:1$ ratio, usually with $4\text{--}7$ carbons. Used for DNA structure, energy, and cell walls.
• Proteins: Composed of amino acids linked by peptide bonds; basic building blocks of the cell.
• Nucleic Acids: Genetic information carriers. DNA acts as the library; RNA is the transcript being read. Composed of $4/5$ types of nucleotides.

Prokaryotic vs. Eukaryotic Classification

• Prokaryotes: Generally smaller and lacking internal membrane systems (no nucleus).
• Phylogenetic Groups:
      * Monophyletic: Includes all descendants of the most recent common ancestor.
      * Paraphyletic: Includes the last common ancestor but only some descendants (e.g., Prokaryotes).
      * Polyphyletic: A group of organisms that do not share a recent common ancestor.
• Bacterial Morphology:
      * Cocci: Spheres.
      * Bacilli: Rods.
      * Haloquadratum: Unique square-shaped bacteria.
• Surface Area to Volume Ratio: As a cell grows larger, the ratio decreases. Small cells have a higher Surface Area to Volume ratio, allowing for greater nutrient exchange and faster growth rates.

Bacterial Cell Structures and Functions

• Plasma Membrane: Selectively permeable barrier; site of nutrient/waste transport and energy conservation.
• Gas Vacuole: Provides buoyancy in aquatic environments.
• Ribosomes: Sites of protein synthesis. Bacterial/Archaeal are $70s$; Eukaryotic are $80s$.
• Inclusions: Storage for Carbon, Phosphorus, and other substances.
• Nucleoid: Region containing DNA; typically not membrane-bound.
• Periplasmic Space:
      * Gram (+): Small or absent.
      * Gram (-): Contains enzymes and binding proteins for nutrient processing.
• Cell Wall: Provides protection; maintains shape and osmotic balance.
      * Peptidoglycan: Mesh-like layer of alternating $NAG$ and $NAM$ subunits. $NAM$ connects to amino acid side chains.
      * D-amino acids: Rare configuration in peptidoglycan that protects against degradation and immune targeting.
• Capsule/Slime Layer: Allows attachment to surfaces.
• Fimbriae and Pili: Surface attachment.
• Flagella: Structures for swimming and swarming motility. Consist of a Filament, Hook, and Basal Body (rotary motor).
• Endospore: Dormant, tough structures produced by some bacteria to survive extreme stress (heat, radiation, desiccation).

Membrane Lipids and Transport Mechanisms

• Hopanoids: Bacterial lipids similar to eukaryotic sterols. They modify membrane properties, are more hydrophobic than phospholipids, and act as platforms for protein complex assembly.
• Transport Types:
      * Passive Diffusion: Small molecules moving from high to low concentration (e.g., Osmosis).
      * Facilitated Diffusion: High to low concentration via transport proteins (e.g., Glucose transport).
      * Active Transport: Low to high concentration requiring energy.
          1. Primary: Uses ATP directly (uniporters); e.g., sodium-potassium pump.
          2. Secondary: Uses ion gradients (cotransport). Includes Symporters (same direction) and Antiporters (opposite directions). E.g., glucose uptake in renal tubules.
          3. Group Translocation: Molecule is chemically modified (e.g., phosphorylated) as it enters. E.g., PEP phosphotransferase system in $E.\,coli$.
      * Endocytosis: Eukaryotes only; invagination of membrane to form vacuoles (e.g., phagocytosis by macrophages).

Cell Wall Specifics: Gram (+) vs. Gram (-)

• Gram (+): Thick peptidoglycan with perpendicular Teichoic Acids.
• Gram (-): Thin peptidoglycan and an outer membrane containing LPS (Lipopolysaccharide).
      * LPS Structure:
          1. O side chain (O antigen): Evokes immune response.
          2. Core Polysaccharide: Negative charges interact with $Ca^{2+}$ to stabilize the membrane and exclude small molecules.
          3. Lipid A: Functions as an endotoxin; can cause septic shock if in the bloodstream.
• Enzymes and Antibiotics:
      * Lysozyme: Breaks bonds between $NAG$ and $NAM$; degrades existing peptidoglycan (Gram +).
      * Penicillin: Inhibits synthesis of peptidoglycan; only effective on growing cells.

Motility and Taxis

• Flagellar Patterns: Monotrichous (one flagellum), Polar (at end), Amphitrichous (one at each end), Lophotrichous (cluster at ends), Peritrichous (entire surface).
• Movement Mechanism:
      * Runs: Counter-clockwise (CCW) rotation.
      * Tumbles: Clockwise (CW) rotation.
      * Directed Movement: In the presence of an attractant, bacteria increase runs and decrease tumbles.
• Taxis Types: Chemotaxis (chemicals), Phototaxis (light), Aerotaxis (oxygen), Osmotaxis (osmotic pressure), Hydrotaxis (water).

Archaea and Domain Comparisons

• Archaeal Features:
      * Methanogens: Reduce $CO_2$ to $CH_4$.
      * Similar to Bacteria: Size, prokaryotic, lack organelles/nucleus.
      * Similar to Eukaryotes: RNA structure, gene-encoding proteins, ribosomes, lack of peptidoglycan.
      * Cell Envelope: Often possess an S-layer (protein/glycoprotein). Flagella are thinner, not hollow, and grow from the base.
      * Bacteriorhodopsin: Protein pump that captures light to move protons out of the cell.

Eukaryotic Features and Endosymbiosis

• Membrane: Contains cholesterol and microdomains; lacks a cell wall.
• Lysosomes: Maintain acidic environment to digest waste via intracellular digestion.
• Nucleus Theories:
      * Karyogenic Hypothesis: Engulfment of a guest prokaryote.
• Endosymbiosis: Mitochondria and chloroplasts were originally bacteria. Evidence includes their independent DNA, ribosomes, reproduction, size, and function.
      * Recent Example: Symbiotic relationship between Pea Aphids and $B.\,aphidicola$.

Virology and Viral Dynamics

• Viral Characteristics: Contain DNA or RNA (not both), replicate only in host cells, lack energy genes, no binary fission.
• Structure: Capsid (protein shell), Nucleocapsid (capsid + genome), Capsomere (subunit).
• Antigenic Changes:
      * Drift: Minor changes (year to year).
      * Shift: Major changes (pandemics).
• Surface Proteins:
      * Hemagglutinin: Binds host cell.
      * Neuraminidase: Facilitates viral exit.
• Replication Steps: 1. Attachment, 2. Entry, 3. Uncoating, 4. Synthesis, 5. Assembly, 6. Release.
• Life Cycles:
      * Lytic: Results in host cell lysis (Virulent phages).
      * Lysogenic: Genome integrates into host genome.
      * Human Cycles: Acute (Ebola), Latent/Chronic (Mono), Carcinogenic (HPV/Cancer).
• Viral Quantification:
      * Direct: Counting particles.
      * Indirect: Hemagglutinin assay, Plaque assay.
      * LD50: Concentration required to kill $50\%$ of infected animals.
• Specific Viruses:
      * Influenza: Enveloped; killed $50\text{--}100\,\text{million}$ people.
      * Polio: Non-enveloped, (+) stranded RNA; $1\%$ cause paralysis.
      * HIV: Binds $CD4$ and $CCR5$ receptor. A delta-32 ccr5 mutation confers immunity.
• Prions: Infectious misfolded proteins causing neurological disease. Categories: Spontaneous, Acquired, Inherited, Sporadic.

Microbial Growth and Reproduction

• Biofilm: Community of bacteria in a protective polysaccharide matrix.
• Cell Division: Mentored by septation and the Z-ring.
      * MreB: Essential for rod-shape; without it, cells become cocci.
      * Crescentin: Gives Vibrio its crescent shape.
• Culture Media:
      * Defined: Known chemical composition.
      * Complex: Unknown exact composition; nutrient-rich.
      * Selective: Favors specific organisms (e.g., MacConkey for Gram -).
      * Differential: Distinguishes based on growth characteristics (e.g., Blood Agar).
• Growth Phases:
      * Lag: Component synthesis.
      * Exponential: Maximal growth rate; uniform population.
      * Stationary: Growth equals death ($\Delta\,\text{Pop} = 0$). Nutrients limited.
      * Death: Death rate exceeds growth.
• Mathematics of Growth: $P = P_o \times 2^n$. $E.\,coli$ doubles in $20\,\text{minutes}$ by starting new DNA synthesis before the previous round finishes.
• Measurement Methods:
      * Microscopic counts (cannot distinguish live/dead easily).
      * Flow cytometer (expensive).
      * Viable counts (serial dilution; counts only living cells).
      * Turbidimetric (light scattering; bad for clumping).

Extremophiles and Environmental Adaptations

• Terminologies:
      * -philic: Requires condition.
      * -tolerant: Survives but does not prefer.
• Salinity: Halophiles pump in inorganic ions or make organic solutes to balance hypertonic environments.
• Temperature:
      * Psychrophiles: Optima $15^{\circ}C$ or lower. Use cold-shock proteins and unsaturated lipids.
      * Thermophiles: Optima $55\text{--}65^{\circ}C$. Use heat-shock proteins, reverse DNA gyrase, and high GC content.
• Oxygen Requirements: Aerobes (need $O_2$), Anaerobes (killed by $O_2$), Facultative (either), Aerotolerant (tolerate but don't use), Microaerophiles (low $O_2$ only).
• Detoxification Enzymes: Catalase, peroxidase, superoxide dismutase to handle Reactive Oxygen Species.

Antimicrobial Therapy and Resistance

• Selective Toxicity: Goal is for high Therapeutic Index ($\frac{\text{toxic dose}}{\text{therapeutic dose}}$).
• Spectrum: Broad vs Narrow spectrum.
• Modes of Action:
      * Bacteriostatic: Inhibits growth (e.g., Tetracycline).
      * Bactericidal: Kills cells (e.g., Aminoglycosides).
• Specific Antibiotics:
      * Penicillin/Cephalosporin: Cell wall synthesis inhibitors ($\beta\text{-lactam}$ ring).
      * Vancomycin: Inhibits transpeptidation by binding to substrate.
      * Tetracycline: Binds $30S$ subunit, prevents tRNA binding.
      * Macrolides: Binds $50S$ subunit, inhibits elongation.
      * Sulfonamides/Trimethoprim: Metabolic antagonists; block folic acid synthesis.
      * Isoniazid: Targets mycolic acid in Tuberculosis (TB).
• Antivirals: Protease inhibitors, Fusion inhibitors, Nucleoside analogs, Neuraminidase inhibitors (e.g., Tamiflu).
• MIC/MLC: Minimum Inhibitory/Lethal Concentration.
• Kirby Bauer Test: Uses zones of clearance to determine antibiotic sensitivity.

Metabolism and Bioenergetics

• Gibbs Free Energy: $\Delta G = \Delta H - T\Delta S$.
      * Exergonic: $\Delta G < 0$ (Spontaneous).     * Endergonic: $\Delta G > 0$ (Non-spontaneous).
• Redox Tower: More negative $E$ are donors; more positive are acceptors.
• Energy math: $\Delta G_0 = -nF\Delta E_0$.
• Enzymes: Lower activation energy. Reached saturation when all active sites are bound. Controlled via Allostery or Covalent Modification.
• Glycolysis:
      * Embden-Meyerhof: Net $2\,\text{ATP}$, $2\,\text{NADH}$, $2\,\text{Pyruvate}$.
      * Entner-Doudoroff: Net $1\,\text{ATP}$, $1\,\text{NADPH}$, $1\,\text{NADH}$, $2\,\text{Pyruvate}$.
• TCA Cycle: For each Acetyl-CoA ($2$ per glucose), produces $2\,CO_2$, $3\,NADH$, $1\,FADH_2$, and $1\,GTP/ATP$.
• ETC: Maximum theoretical yield of aerobic respiration is $32\text{--}38\,ATP$, though actual yield is closer to $30$.
• Special Metabolism:
      * Chemolithotrophy: Energy from inorganic molecules.
      * Phototrophy vs. Photosynthesis: Phototrophy is using light for energy; Photosynthesis involves carbon fixation.
      * Fermentation: No external electron acceptor; uses organic compounds.

Molecular Genetics and Information Flow

• Experiments:
      * Griffith: Transforming principle in smooth/rough strains.
      * McCarthy/MacLeod: Proved DNA is the transforming molecule using DNase.
      * Hershey/Chase: Used radioactive Phosphorus ($P$) and Sulfur ($S$) to confirm DNA enters cells via bacteriophages.
• Replication: Semiconservative. Includes DNA Pol III (synthesis), Primase (RNA primer), Gyrase (tension relief), and Helicase (unwinding).
• Transcription: Uses Sigma factors to help RNA polymerase find promoter sites. Polycistronic mRNA allows multiple genes on one transcript.
• Translation: 61 codons (plus 3 stop codons). Uses $A$ (entry), $P$ (peptide bond), and $E$ (exit) sites. Bacteria use N-formylmethionine as the start amino acid.
• Regulation:
      * Negative Control: Induction/Repression (transcription happens unless blocked).
      * Positive Control: Transcription requires an inducer/activator.
      * Quorum Sensing: Bacterial density assessment (e.g., Lux pathway). High cell density inhibits the sensor kinase that makes sRNA, freeing the operon.
      * Attenuation: Translation limits transcription of Tryptophan based on amino acid availability.

Genetic Variance and Horizontal Gene Transfer

• Mutations:
      * Point Mutations: Missense, Nonsense (stop), Silent.
      * Indel: Insertion/deletion leading to Frameshifts.
      * Auxotrophs: Mutants requiring additional nutrients; identified via Replica Plating.
      * Ames Test: Used to evaluate mutagens.
• Horizontal (Lateral) Gene Transfer:
      * Conjugation: Cell-to-cell mating via sex pilus; requires an $F\text{-plasmid}$. Hfr cells have the plasmid integrated into the chromosome.
      * Transformation: Uptake of naked DNA.
      * Transduction: DNA transfer via viruses (Generalized vs. Specialized).

Taxonomy and Biogeochemical Cycles

• Microbial Species: Defined by Genetic Species Concept (>70\,\% DNA hybridization and >97\,\% $16S\,rRNA$ identity).
• OTU: Operational Taxonomic Unit; the functional equivalent of species based on GSC data.
• Nitrogen Cycle: Nitogen ($N_2$) is $78\,\%$ of atmosphere but not bioavailable. Peanuts have a symbiotic relationship with Rhizobiales to fix $N_2$ into $NH_3$.
• Eutrophication: Over-fertilization ($N_2$ runoff) leading to algae blooms.
• Oxygenation: Rise of $O_2$ occurred $2.4\,\text{Gya}$ after oxygenic photosynthesis evolved in Cyanobacteria (>2.7\,\text{Gya}).

Host-Microbe Interactions and Immunity

• Infection Steps: Exposure, Adherence, Colonization/Growth, Virulence Factor production.
• Toxins:
      * Exotoxins: Released proteins.
          * AB Toxins: B subunit binds, A subunit damages (e.g., Diphtheria inhibits protein synthesis; Cholera creates extra cAMP and diarrhea).
          * Botulism: Blocks ACh; causes relaxation (lethal).
          * Tetanus: Blocks glycine; causes contraction.
          * Superantigens: Overstimulate T-cells and cytokines.
      * Endotoxins: Found in the LPS of Gram (-) bacteria.
• Immune System: Adaptive response features specificity, memory, and tolerance.
• Vaccines:
      * Attenuated: Live, weakened (MMR). Best immunity.
      * Inactivated: Killed (Flu). Safer but weaker.
      * Toxoid: Inactivated toxins (Tetanus).
• Human Microbiome: Ratio of microbial to human cells is $10:1$. Vaginal birth promotes Lactobacillus; C-section results in Staphylococcus increase. The microbiome aids in vitamin production and calorie extraction ($20\,\%$ lipid extraction increase).