Microbiology Exam Study Guide: Metabolism, Genetics, and Gene Transfer

Lecture 7: Bioenergetics and Introduction to Metabolism

Fundamental Requirements for Cellular Life

All living cells require certain basic elements and conditions to sustain life and carry out metabolic processes.

• Water: Essential solvent and medium for biochemical reactions.

• Nutrients: Include carbon, nitrogen, phosphorus, sulfur, and trace elements.

• Free Energy: Energy available to do cellular work.

• Reducing Power: Molecules like NADH and FADH2 that donate electrons in metabolic reactions.

Free Energy in Cellular Life

Free energy is the energy available to do work in a cell. It is used in chemical, transport, and mechanical work.

• Chemical Work: Synthesis of macromolecules.

• Transport Work: Movement of substances across membranes.

• Mechanical Work: Movement of cell structures.

Thermodynamics in Metabolism

Thermodynamics governs the energy changes in metabolic reactions.

• First Law: Energy cannot be created or destroyed, only transformed.

• Second Law: Entropy (disorder) increases in spontaneous processes.

Change in Free Energy and Reaction Direction

The change in free energy ($\Delta G$) determines whether a reaction will proceed spontaneously.

• Negative $\Delta G$: Reaction releases energy (exergonic), proceeds spontaneously.

• Positive $\Delta G$: Reaction requires energy input (endergonic), non-spontaneous.

• Standard Free Energy Change ($\Delta G^\circ'$): Calculated under standard conditions.

Equilibrium and Reaction Prediction

The value of $\Delta G$ can be used to predict the direction of metabolic reactions and their equilibrium.

• Equilibrium Constant ($K_{eq}$): Relates to $\Delta G$ by $\Delta G = -RT \ln K_{eq}$.

• Spontaneity: If $K_{eq} > 1$, $\Delta G$ is negative; reaction favors products.

Types of Metabolism

Metabolism is divided into two main types:

• Catabolism: Breakdown of molecules to release energy.

• Anabolism: Synthesis of complex molecules from simpler ones.

ATP and Energy Conservation

ATP (adenosine triphosphate) is the universal energy currency in cells.

• ATP Structure: Adenine base, ribose sugar, three phosphate groups.

• High-Energy Bonds: Phosphoanhydride bonds between phosphate groups store energy.

• ATP Hydrolysis: Releases energy for cellular work.

ATP Synthesis Mechanisms

• Substrate-Level Phosphorylation: Direct transfer of phosphate to ADP during glycolysis and TCA cycle.

• Oxidative Phosphorylation: ATP generated by electron transport chain using energy from redox reactions.

• Photophosphorylation: ATP synthesis using light energy (in phototrophs).

Redox Reactions and Electron Carriers

Redox reactions involve the transfer of electrons between molecules, crucial for energy production.

• Reduction Potential ($E_0'$): Tendency of a compound to accept electrons; measured in volts.

• Electron Carriers: NAD+/NADH, FAD/FADH2, cytochromes.

• Electron Flow: Electrons move from carriers with lower reduction potential to those with higher potential.

General Aspects of Metabolism

• Metabolic pathways are highly regulated and interconnected.

• Energy conservation and electron flow are central to all cellular life.

Lecture 8: Microbial Metabolism 1

Metabolic Classes of Microorganisms

Microorganisms are classified based on their energy and carbon sources.

• Phototrophs: Use light as energy source.

• Chemotrophs: Use chemicals as energy source.

• Chemoorganotrophs: Use organic compounds.

• Chemolithotrophs: Use inorganic compounds.

Glycolysis Pathway (Embden-Meyerhof-Parnas)

Glycolysis is the central pathway for glucose catabolism.

• Substrate: Glucose (6 carbons).

• End Products: Pyruvate, ATP, NADH.

• ATP Yield: Net 2 ATP per glucose (substrate-level phosphorylation).

• NADH Yield: 2 NADH per glucose.

• Preparatory Phase: Consumes 2 ATP.

• Energy-Conserving Phase: Produces 4 ATP.

• Fermentative vs. Aerobic Glycolysis: Fermentative microbes regenerate NAD+ by reducing pyruvate.

Catabolic Fate of Pyruvate

• Aerobic Respiration: Pyruvate enters TCA cycle and ETC.

• Anaerobic Respiration: Pyruvate reduced by alternative electron acceptors.

• Fermentation: Pyruvate reduced to regenerate NAD+.

Citric Acid Cycle (TCA/Krebs Cycle)

The TCA cycle oxidizes acetyl-CoA to CO2 and generates energy carriers.

• Entry: Acetyl-CoA combines with oxaloacetate.

• CO2 Release: Two molecules per cycle.

• Energy Carriers: NADH, FADH2, GTP/ATP.

• Net Yield: 3 NADH, 1 FADH2, 1 GTP/ATP per cycle.

Electron Transport Chain (ETC)

ETC transfers electrons from NADH/FADH2 to final electron acceptors, generating a proton motive force (PMF).

• Components: Complexes I-IV, cytochromes, quinones.

• Location: Bacterial cell membrane, mitochondrial inner membrane in eukaryotes.

• PMF: Drives ATP synthesis via ATP synthase.

Oxidative Phosphorylation

• ATP synthesis coupled to electron transport and PMF.

• Higher ATP yield when O2 is the final electron acceptor.

Lecture 9: Microbial Metabolism 2

Fermentation vs. Respiration

Microbes use different strategies to generate ATP depending on the availability of electron acceptors.

• Fermentation: ATP generated by substrate-level phosphorylation; organic molecules are electron acceptors.

• Respiration: ATP generated by oxidative phosphorylation; inorganic molecules (e.g., O2) are electron acceptors.

• ATP Yield: Aerobic respiration yields more ATP than fermentation.

Carbon Sources for Biosynthesis

• Organic Source: Fixed carbon from catabolic intermediates.

• Inorganic Source: CO2 fixation by autotrophs.

Anabolic Pathways

• Biosynthesis: Pathways that build macromolecules from simple precursors.

• Autotrophs: Fix carbon from CO2.

• Heterotrophs: Use organic carbon sources.

Calvin Cycle

The Calvin cycle is the main pathway for CO2 fixation in autotrophs.

• Phases: Carboxylation, reduction, regeneration.

• ATP/NADPH Use: Consumes ATP and NADPH to fix CO2 into sugars.

Reverse TCA Cycle

• Some bacteria use the reverse TCA cycle to fix CO2 and generate precursors for biosynthesis.

Pentose Phosphate Pathway

• Generates NADPH and pentoses for anabolic reactions.

Nitrogen Assimilation

• Sources: Ammonia (NH3), nitrate (NO3-), nitrogen gas (N2).

• Pathways: Nitrogen fixation, assimilation, and reduction.

Amino Acid and Carbon Skeleton Biosynthesis

• Reductive Amination: Incorporation of ammonia into carbon skeletons.

• Glutamine Synthetase: Key enzyme for nitrogen assimilation.

Glyoxylate Cycle

• Bypasses decarboxylation steps of TCA cycle, allowing growth on acetate or fatty acids.

Lecture 11: Microbial Genetics and Replication

Genetics Vocabulary

• DNA: Deoxyribonucleic acid, genetic material.

• RNA: Ribonucleic acid, involved in gene expression.

• Gene: Sequence of DNA encoding a functional product.

• Nucleotides: Building blocks of DNA/RNA.

• dNTPs: Deoxynucleotide triphosphates (DNA synthesis).

• NTPs: Nucleotide triphosphates (RNA synthesis).

DNA Structure

• Pentose Sugar: Deoxyribose in DNA, ribose in RNA.

• Phosphate Group: Attached to 5' carbon.

• Nitrogenous Base: Attached to 1' carbon; A, T, G, C (U in RNA).

• Phosphodiester Bond: Links 5' phosphate to 3' OH of adjacent nucleotide.

Bases in DNA

• G-C: 3 hydrogen bonds (higher melting point).

• A-T: 2 hydrogen bonds.

• RNA: U replaces T.

Double-Stranded DNA Structure

• Antiparallel strands, right-handed helix.

• Major and minor grooves for protein binding.

Supercoiling and DNA Gyrase

• Supercoiling compacts DNA inside cells.

• DNA gyrase introduces negative supercoils.

• Microbial DNA is circular; eukaryotic DNA is linear.

Central Dogma and Molecular Information Flow

• Replication: DNA synthesis.

• Transcription: DNA to RNA.

• Translation: RNA to protein.

DNA Replication in Bacteria

• Semiconservative: Each new DNA has one old and one new strand.

• Origin of Replication (OriC): Starting point.

• Helicase: Unwinds DNA.

• DNA Polymerases: Pol I, Pol III (major for synthesis).

• Primase: Synthesizes RNA primers.

• Leading Strand: Continuous synthesis.

• Lagging Strand: Discontinuous synthesis (Okazaki fragments).

• DNA Ligase: Seals nicks between fragments.

Polymerase Chain Reaction (PCR)

• Invented by Kary Mullis.

• Uses temperature cycles for DNA denaturation, annealing, and extension.

• Taq polymerase from thermophilic microbes enables automation.

Lecture 12: Horizontal Gene Transfer (HGT)

Mechanisms of HGT

• Transformation: Uptake of free DNA from environment.

• Transduction: DNA transfer via bacteriophages.

• Conjugation: Direct transfer between cells via pilus.

Mobile Genetic Elements (MGEs)

• Transposons, insertion sequences, plasmids, bacteriophages.

• Facilitate movement of genes within and between genomes.

Significance of HGT

• Rapid genetic change and adaptation in bacteria.

• Spread of antibiotic resistance.

• Impact on evolution and biotechnology.

Bacteriophages

• Viruses that infect bacteria.

• Lytic phages cause cell lysis; lysogenic phages integrate into host genome.

HGT in the Environment

• Occurs in diverse habitats: soil, water, human gut, hospitals, agriculture.

Lecture 13: Bacterial Transcription and Translation

Transcription

• Genetic information in DNA is transcribed to mRNA.

• RNA polymerase binds to promoter regions (e.g., -35 and -10 sequences).

• Sigma factor enables RNA polymerase binding.

• Termination can be rho-independent or rho-dependent.

Operons and mRNA Types

• Monocistronic mRNA: Encodes one protein.

• Polycistronic mRNA: Encodes multiple proteins (common in bacteria).

• Operon: Cluster of genes under control of a single promoter.

Translation

• mRNA is translated into polypeptides by ribosomes.

• Genetic Code: 64 codons (61 sense, 3 stop).

• Start Codon: AUG (methionine).

• Stop Codons: UAA, UAG, UGA.

• tRNA: Cloverleaf structure, anticodon matches mRNA codon.

• Ribosome: 70S (prokaryotes), composed of 50S and 30S subunits.

Protein Secretion Systems

• Sec System: Translocates proteins across the cytoplasmic membrane.

• Tat System: Transports folded proteins.

Additional info:

• For equations: $\Delta G = -RT \ln K_{eq}$ (relates free energy change to equilibrium constant).

• ATP hydrolysis: $ATP + H_2O \rightarrow ADP + P_i + \text{energy}$

• DNA replication direction: 5' to 3'.