🦠Microbiology Unit 7 – Microbial Biochemistry

Key Concepts and Terminology

• Metabolism encompasses all chemical reactions involved in maintaining the living state of cells and organisms
• Anabolism constructs molecules from smaller units (requires energy input)
• Catabolism breaks down molecules into smaller units (releases energy)
• Enzymes act as biological catalysts accelerating chemical reactions without being consumed in the process
• Cofactors are non-protein compounds required for enzyme activity (includes coenzymes and metal ions)
• Substrates are molecules acted upon by enzymes in a biochemical reaction
• Metabolic pathways are series of enzymatic reactions that transform initial substrates into final products
  • Linear pathways have a single start and end point
  • Branched pathways have multiple start or end points
  • Cyclic pathways have the end product regenerate the starting molecule

Microbial Cell Structure

• Prokaryotic cells (bacteria and archaea) lack membrane-bound organelles and a true nucleus
• Cell wall provides structural support and protection (peptidoglycan in bacteria, pseudopeptidoglycan in archaea)
• Plasma membrane is a selective barrier controlling the passage of substances in and out of the cell
  • Composed of phospholipid bilayer with embedded proteins
  • Fluid mosaic model describes the dynamic nature of the membrane
• Cytoplasm contains enzymes, ribosomes, and other cellular components
• Nucleoid is a region containing the cell's genetic material (DNA) without a nuclear membrane
• Plasmids are small, circular DNA molecules that replicate independently of the main chromosome
• Ribosomes are the site of protein synthesis (70S in prokaryotes, consisting of 50S and 30S subunits)
• Inclusions are intracellular storage granules (glycogen, polyphosphate, sulfur)

Metabolic Pathways in Microbes

• Glycolysis is a central pathway that oxidizes glucose to pyruvate (occurs in the cytoplasm)
• Citric acid cycle (Krebs cycle) oxidizes acetyl-CoA to CO2, generating high-energy compounds (NADH, FADH2)
• Pentose phosphate pathway produces NADPH and pentose sugars for biosynthesis (ribose for nucleotides)
• Entner-Doudoroff pathway is an alternative to glycolysis in some bacteria (Pseudomonas)
• Glyoxylate cycle allows the use of acetate or fatty acids as sole carbon sources (bypasses CO2-generating steps of Krebs cycle)
• Fermentation pathways regenerate NAD+ in the absence of oxygen (lactic acid, ethanol, mixed acid fermentation)
• Amino acid catabolism provides carbon skeletons for energy production and biosynthesis
  • Transamination transfers amino group to α-ketoglutarate, forming glutamate
  • Deamination removes amino group as ammonia (NH3)

Enzymes and Catalysis

• Active site is the region of an enzyme where the substrate binds and the reaction occurs
• Induced fit model suggests that substrate binding causes conformational changes in the enzyme
• Michaelis-Menten kinetics describes the relationship between substrate concentration and reaction rate
  • $V_0 = \frac{V_{max}[S]}{K_m + [S]}$
  • $V_0$ is the initial reaction rate, $V_{max}$ is the maximum rate, $[S]$ is substrate concentration, and $K_m$ is the Michaelis constant
• Competitive inhibition occurs when an inhibitor competes with the substrate for the active site
• Non-competitive inhibition occurs when an inhibitor binds to a site other than the active site, altering enzyme conformation
• Allosteric regulation involves the binding of effectors at sites other than the active site, modulating enzyme activity
• Feedback inhibition is a form of allosteric regulation where the end product of a pathway inhibits the first enzyme in the pathway
• Enzyme activity can be affected by temperature, pH, and substrate concentration

Energy Production in Microorganisms

• Electron transport chain (ETC) is a series of redox reactions that generate a proton gradient across the membrane
  • Electrons from reduced compounds (NADH, FADH2) are transferred through a series of carriers (cytochromes)
  • Protons are pumped across the membrane, creating an electrochemical gradient
• Chemiosmosis couples the proton gradient to ATP synthesis via ATP synthase
• Substrate-level phosphorylation directly produces ATP through the transfer of a phosphate group to ADP (glycolysis, Krebs cycle)
• Photophosphorylation generates ATP using light energy in phototrophs (purple bacteria, cyanobacteria)
  • Cyclic photophosphorylation involves a single photosystem (PS I) and generates ATP
  • Noncyclic photophosphorylation involves both PS I and PS II, generating ATP and NADPH
• Oxidative phosphorylation refers to the combined process of electron transport and chemiosmosis
• Aerobic respiration uses oxygen as the final electron acceptor, yielding the most ATP per glucose molecule
• Anaerobic respiration uses alternative electron acceptors (nitrate, sulfate, iron) in the absence of oxygen

Biosynthesis and Growth

• Anabolic pathways synthesize complex molecules from simpler precursors
• Amino acid biosynthesis involves the incorporation of nitrogen into carbon skeletons
  • Glutamate and glutamine are central to nitrogen metabolism
  • Transamination reactions transfer amino groups to α-ketoacids
• Nucleotide biosynthesis produces purines (ATP, GTP) and pyrimidines (CTP, UTP, TTP)
  • Ribonucleotides are synthesized first, then reduced to deoxyribonucleotides
• Fatty acid biosynthesis occurs through the repeated addition of two-carbon units (acetyl-CoA)
  • Fatty acid synthase is a multienzyme complex that catalyzes the reactions
• Lipid biosynthesis incorporates fatty acids into phospholipids and other complex lipids
• Peptidoglycan biosynthesis involves the assembly of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) units
• Growth curve represents the phases of microbial growth in a closed system (lag, exponential, stationary, death)
  • Exponential (log) phase is characterized by a constant doubling time

Regulation of Microbial Metabolism

• Catabolite repression is the preferential use of a readily available carbon source (glucose) over others
  • Lac operon in E. coli is a classic example of catabolite repression and induction
• Transcriptional regulation controls gene expression at the level of mRNA synthesis
  • Repressors bind to operators, preventing transcription (negative control)
  • Activators bind to enhancers, promoting transcription (positive control)
• Attenuation is a form of transcriptional regulation that involves premature termination of transcription
  • Trp operon in E. coli is regulated by attenuation in response to tryptophan levels
• Post-transcriptional regulation controls gene expression after mRNA synthesis
  • Riboswitches are mRNA sequences that bind specific metabolites, altering mRNA stability or translation
• Translational regulation controls gene expression at the level of protein synthesis
  • Shine-Dalgarno sequence is a ribosome binding site that affects translation initiation
• Post-translational modifications (phosphorylation, glycosylation) can modulate enzyme activity
• Two-component systems allow bacteria to respond to environmental signals
  • Histidine kinase senses the signal and phosphorylates a response regulator, which effects a cellular response

Practical Applications and Lab Techniques

• Isolation of pure cultures is essential for studying microbial metabolism
  • Streak plate method involves sequential dilution of a mixed culture on solid media
  • Pour plate and spread plate methods allow for quantification of viable cells (colony-forming units, CFU)
• Selective and differential media contain specific nutrients or indicators to isolate or identify microbes
  • MacConkey agar selects for gram-negative bacteria and differentiates lactose fermenters (pink colonies)
  • Mannitol salt agar selects for salt-tolerant bacteria and differentiates mannitol fermenters (yellow colonies)
• Biochemical tests aid in the identification of microorganisms based on their metabolic capabilities
  • Catalase test detects the presence of the enzyme catalase, which decomposes hydrogen peroxide
  • Oxidase test detects the presence of cytochrome c oxidase, which is involved in the electron transport chain
• Antimicrobial susceptibility testing determines the sensitivity of a microorganism to specific antibiotics
  • Kirby-Bauer disk diffusion method measures the zone of inhibition around antibiotic-impregnated disks
  • Minimum inhibitory concentration (MIC) is the lowest concentration of an antibiotic that inhibits visible growth
• Metabolic engineering involves the modification of microbial metabolic pathways for the production of desired compounds
  • Recombinant DNA technology allows for the introduction of foreign genes or the alteration of existing pathways
  • Biofuels (ethanol, butanol) and pharmaceuticals (insulin, antibiotics) are examples of products obtained through metabolic engineering