Microbial Metabolism

Oxidation and Reduction Reactions

Oxidation-reduction (redox) reactions are fundamental to microbial metabolism, involving the transfer of electrons between molecules.

• Oxidation: Loss of electrons from a molecule.

• Reduction: Gain of electrons by a molecule.

• Example: In cellular respiration, glucose is oxidized to carbon dioxide, while oxygen is reduced to water.

Phosphorylation Mechanisms

Phosphorylation is the addition of a phosphate group to a molecule, crucial for ATP synthesis.

• Substrate-level phosphorylation: Direct transfer of a phosphate group to ADP from a phosphorylated intermediate. Occurs in glycolysis and the Krebs cycle.

• Oxidative phosphorylation: ATP is generated from the transfer of electrons through the electron transport chain (ETC) to oxygen, creating a proton gradient used by ATP synthase. Occurs in the inner mitochondrial membrane (eukaryotes) or plasma membrane (prokaryotes).

• Photophosphorylation: Light energy is used to generate ATP via an ETC in photosynthetic organisms. Occurs in thylakoid membranes (eukaryotes) or cytoplasmic membrane (prokaryotes).

Enzymes in Metabolism

Enzymes are biological catalysts made of proteins (and sometimes RNA) that speed up metabolic reactions.

• Substrate: The molecule upon which an enzyme acts.

• Active site: The region on the enzyme where the substrate binds.

• Competitive regulation: Inhibitor competes with substrate for the active site.

• Allosteric regulation: Inhibitor or activator binds to a site other than the active site, changing enzyme activity.

• Apoenzyme: The protein portion of an enzyme, inactive without its cofactor.

• Holoenzyme: The complete, active enzyme with its cofactor.

• Ribozyme: RNA molecules with catalytic activity.

Cellular Respiration

Cellular respiration is the process by which cells extract energy from organic molecules.

• Glycolysis: Occurs in the cytoplasm; converts glucose to 2 pyruvate, producing 2 ATP (substrate-level), 2 NADH.

• Formation of Acetyl-CoA: Pyruvate is converted to acetyl-CoA, producing 2 NADH and 2 CO2 per glucose.

• Krebs (Citric Acid) Cycle: Occurs in the mitochondrial matrix (eukaryotes) or cytoplasm (prokaryotes); produces 2 ATP (substrate-level), 6 NADH, 2 FADH2, and 4 CO2 per glucose.

• Electron Transport Chain (ETC): Located in the inner mitochondrial membrane (eukaryotes) or plasma membrane (prokaryotes); uses NADH and FADH2 to generate a proton gradient for ATP synthesis (oxidative phosphorylation).

ATP Yield Table:

Additional info: Actual ATP yield may vary depending on organism and conditions.

Terminal Electron Acceptors

• Aerobic respiration: Oxygen is the terminal electron acceptor; yields the most ATP (~38 per glucose in prokaryotes).

• Anaerobic respiration: Inorganic molecules other than oxygen (e.g., nitrate, sulfate) serve as terminal electron acceptors; yields less ATP.

• Fermentation: Organic molecules (e.g., pyruvate) are the terminal electron acceptors; yields 2 ATP per glucose.

Fermentation

Fermentation is an anaerobic process that regenerates NAD+ for glycolysis by reducing pyruvate or its derivatives.

• Purpose: To allow glycolysis to continue in the absence of oxygen.

• ATP yield: 2 ATP per glucose (from glycolysis only).

• Major pathways:

  • Lactic acid fermentation: Produces lactic acid (e.g., in muscle cells, Lactobacillus).

  • Alcoholic fermentation: Produces ethanol and CO2 (e.g., in yeast).

Photosynthesis: Light-Dependent Reactions

Light-dependent reactions convert light energy into chemical energy (ATP and NADPH).

• Location: Thylakoid membranes (eukaryotes), cytoplasmic membrane (prokaryotes).

• Water: Split to provide electrons, releasing O2.

• ATP generation: Via photophosphorylation using ATP synthase.

• Photosystems: Photosystem II and I participate in electron flow.

• NADPH: Generated via Photosystem I.

• Terminal electron acceptor: NADP+ in the light reactions.

Photosynthesis: Light-Independent Reactions (Calvin Cycle)

The Calvin cycle uses ATP and NADPH to fix CO2 into organic molecules.

• Three steps: Carbon fixation, reduction, regeneration of RuBP.

• Product: Glyceraldehyde-3-phosphate (G3P); two G3P are needed to make one glucose.

• Turns per glucose: Six turns of the cycle are required to produce enough G3P for one glucose.

Photorespiration

Photorespiration is a wasteful process where Rubisco fixes O2 instead of CO2, reducing photosynthetic efficiency, especially under high O2 and low CO2 conditions.

Microbial Genetics

DNA Structure

DNA is a double helix composed of nucleotides.

• Phosphate-sugar backbone: Phosphodiester bonds link phosphate to sugar.

• Sugar-base: Covalent (glycosidic) bonds connect sugar to base.

• Base pairs: Hydrogen bonds hold bases together (A-T: 2 bonds, G-C: 3 bonds).

• Bases: Adenine (A), Thymine (T), Cytosine (C), Guanine (G).

DNA Polarity and Antiparallel Structure

• 5’ and 3’ ends: Refer to the carbon positions on the deoxyribose sugar; DNA strands run in opposite directions (antiparallel).

Organization of Prokaryotic vs Eukaryotic DNA

• Prokaryotes: Circular DNA, usually one chromosome, found in nucleoid; may have plasmids.

• Eukaryotes: Linear chromosomes, multiple per cell, found in nucleus; DNA wrapped around histones.

Plasmids

• Definition: Small, circular, double-stranded DNA molecules in prokaryotes.

• Genes: Often carry genes for antibiotic resistance, virulence, or metabolism.

DNA Replication

DNA replication is the process of copying DNA before cell division.

• Enzymes:

  • Helicase: Unwinds DNA.

  • Primase: Synthesizes RNA primers.

  • DNA polymerase: Adds nucleotides to the growing DNA strand.

  • Ligase: Joins Okazaki fragments.

  • Gyrase/topoisomerase: Relieves supercoiling.

• Directionality: New DNA is synthesized 5’ to 3’.

• Semi-conservative: Each new DNA molecule has one old and one new strand.

• Leading strand: Synthesized continuously.

• Lagging strand: Synthesized discontinuously as Okazaki fragments.

Transcription (DNA to mRNA)

• Steps: Initiation (RNA polymerase binds promoter), elongation (RNA synthesis), termination.

• Enzyme: RNA polymerase.

• Promoter: DNA sequence where RNA polymerase binds to start transcription.

• Direction: 5’ to 3’ synthesis; only one DNA strand is transcribed.

• Termination in prokaryotes: Rho-dependent and rho-independent mechanisms.

• RNA processing: Eukaryotes add 5’ cap, poly-A tail, and splice introns; prokaryotes do not.

Translation (mRNA to Protein)

• Steps: Initiation (formation of initiation complex), elongation (polypeptide synthesis), termination (release of polypeptide).

• Required RNAs: mRNA (template), tRNA (brings amino acids), rRNA (structural and catalytic component of ribosome).

• Codon: Three-nucleotide sequence on mRNA coding for an amino acid.

• Anticodon: Complementary three-nucleotide sequence on tRNA.

• Shine-Dalgarno sequence: Ribosome binding site in prokaryotic mRNA.

• Direction: 5’ to 3’ along mRNA.

• Codons: 64 total; 61 code for amino acids, 3 are stop codons, 1 start codon (AUG for methionine).

• Termination: Stop codon is reached; release factors free the polypeptide.

• Ribosome structure: Large and small subunits; rRNA in large subunit catalyzes peptide bond formation.

Operons

• Structure: Promoter, operator, regulatory gene, and structural genes.

• Polycistronic: One mRNA codes for multiple proteins (common in prokaryotes).

• Inducible operons: Usually off, turned on by inducer (e.g., lac operon).

• Repressible operons: Usually on, turned off by corepressor (e.g., trp operon).

• cAMP and CAP: cAMP binds CAP protein, which enhances lac operon transcription when glucose is low.

Mutations

• Point mutations: Single nucleotide changes (e.g., silent, missense, nonsense).

• Silent mutation: No change in amino acid.

• Missense mutation: Changes one amino acid.

• Nonsense mutation: Creates a stop codon.

• Frameshift mutation: Insertion or deletion shifts reading frame.

• Causes: Spontaneous errors, chemicals, radiation.

Horizontal Gene Transfer in Prokaryotes

• Transformation: Uptake of naked DNA from environment.

• Transduction: Transfer by bacteriophages.

• Conjugation: Direct transfer via pilus.

Viruses, Viroids, and Prions

Viral Genomes

• Types: DNA or RNA, single- or double-stranded, linear or circular.

Positive and Negative RNA

• Positive-sense RNA: Can be directly translated by host ribosomes.

• Negative-sense RNA: Must be converted to positive-sense RNA by RNA-dependent RNA polymerase.

Viral Polymerases

• RNA viruses: Require RNA-dependent RNA polymerase for replication.

Viral Envelope

• Origin: Derived from host cell membrane during viral budding.

• Significance: Enveloped viruses are more sensitive to detergents and desiccation; envelope aids in host cell entry and immune evasion.

Lytic Cycle vs Lysogeny

Prophage and Provirus

• Prophage: Bacteriophage DNA integrated into bacterial genome.

• Provirus: Animal virus DNA integrated into host genome.

• Induction: Activation of prophage to enter lytic cycle.

• Difference: Animal proviruses may remain permanently in host DNA; prophages can be excised.

Viroids

• Definition: Small, circular RNA molecules without protein coat.

• Infect: Plants.

Prions

• Definition: Infectious proteins causing neurodegenerative diseases.

• Significance: Lack nucleic acids; resistant to standard sterilization.

• Diseases: Creutzfeldt-Jakob disease, mad cow disease (BSE).

• Treatment: No effective treatment; prevention is key.

• Destruction: Incineration or autoclaving at high temperatures with strong chemicals.