Microbial Metabolism, Growth, Genetics, Control, and Antimicrobial Agents: Study Guide for BIOL 2420 Exam #2

Microbial Metabolism

Catabolism vs. Anabolism

Metabolism encompasses all chemical reactions within a cell, divided into catabolism (breakdown) and anabolism (synthesis). These processes are interconnected, with catabolic reactions providing energy and precursors for anabolic reactions.

• Catabolism: Breakdown of complex molecules into simpler ones, releasing energy (exergonic). Example: Glycolysis.

• Anabolism: Synthesis of complex molecules from simpler ones, requiring energy (endergonic). Example: Protein synthesis.

• Link: Catabolic pathways generate ATP and reducing power (NADH, FADH2) used in anabolic pathways.

Key Definitions

• Metabolism: All chemical reactions in a cell.

• Endergonic: Reactions that require energy input.

• Exergonic: Reactions that release energy.

Amphibolic Reactions

Amphibolic reactions serve both catabolic and anabolic functions, allowing flexibility in metabolic pathways.

• Example: Citric acid cycle intermediates used for biosynthesis.

ATP: Structure and Function

ATP (adenosine triphosphate) is the primary energy carrier in cells.

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

• Function: Stores and transfers energy for cellular processes.

Enzymes: Structure, Function, and Regulation

Enzymes are biological catalysts that speed up reactions by lowering activation energy.

• Structure: Protein (sometimes with cofactors/coenzymes).

• Function: Specific for substrates; catalyze reactions efficiently.

• Denaturation: Loss of enzyme structure and function due to extreme conditions (temperature, pH).

• Activation Energy: Minimum energy required to start a reaction.

• Enzyme Activity Graph: Shows effect of enzyme on activation energy.

Enzyme Inhibition

• Allosteric Inhibition: Inhibitor binds to site other than active site, changing enzyme shape.

• Competitive Inhibition: Inhibitor competes with substrate for active site.

• Noncompetitive Inhibition: Inhibitor binds elsewhere, reducing enzyme activity.

• Feedback Inhibition: End product inhibits pathway, maintaining homeostasis.

• Sulfanilamide: Acts as a competitive inhibitor of folic acid synthesis in bacteria.

Oxidation-Reduction (Redox) Reactions

Redox reactions transfer electrons, crucial for energy production.

• Reducing Power: Molecules like NADH and FADH2 carry electrons.

• Coenzymes: NAD+, FAD, NADP+ are essential for redox reactions.

Carbohydrate Catabolism Pathways

Cells break down carbohydrates through a series of pathways to generate energy.

1. Glycolysis: Occurs in cytoplasm; converts glucose to pyruvate, produces ATP and NADH.

2. Acetyl-CoA Synthesis: Pyruvate converted to acetyl-CoA; produces NADH and CO2.

3. Citric Acid Cycle (Krebs Cycle): Occurs in cytoplasm (prokaryotes) or mitochondria (eukaryotes); produces ATP, NADH, FADH2, CO2.

4. Electron Transport Chain (ETC): Located in plasma membrane (prokaryotes) or mitochondrial membrane (eukaryotes); uses NADH/FADH2 to generate ATP via chemiosmosis.

Summary Table: Catabolic Pathways

Pathway	Location (Prokaryote)	Location (Eukaryote)	Starting Substance	Products
Glycolysis	Cytoplasm	Cytoplasm	Glucose	Pyruvate, ATP, NADH
Acetyl-CoA Synthesis	Cytoplasm	Mitochondria	Pyruvate	Acetyl-CoA, NADH, CO2
Citric Acid Cycle	Cytoplasm	Mitochondria	Acetyl-CoA	CO2, ATP, NADH, FADH2
ETC	Plasma Membrane	Mitochondrial Membrane	NADH, FADH2	ATP, H2O

Chemiosmosis and Proton Movement

Proton movement across membranes during chemiosmosis drives ATP synthesis via ATP synthase.

• Function: Establishes proton gradient; energy used to produce ATP.

Fermentation

Fermentation allows cells to regenerate NAD+ for glycolysis in absence of oxygen.

• Types: Alcoholic (produces ethanol), lactic acid (produces lactate).

• Purpose: Replenishes NAD+ for continued glycolysis.

Catabolism of Fats and Proteins

• Beta-Oxidation: Fatty acids broken down to acetyl-CoA.

• Deamination: Removal of amino group from proteins; enters central catabolic pathways.

• Central Catabolism: Carbohydrate catabolism is central as other macromolecules feed into these pathways.

Phosphorylation Types

• Oxidative Phosphorylation: ATP generated via ETC and chemiosmosis.

• Substrate-Level Phosphorylation: ATP generated directly in metabolic reactions (e.g., glycolysis).

Regulation of Metabolism

• Integration: Cells regulate metabolism via feedback inhibition, gene expression, and enzyme activity.

Microbial Nutrition and Growth

Oxygen Requirements and Energy Metabolism

Microbes are classified by their oxygen requirements, which influence their energy metabolism and protective mechanisms against toxic oxygen.

• Obligate Aerobe: Requires oxygen; aerobic respiration.

• Obligate Anaerobe: Cannot tolerate oxygen; anaerobic respiration or fermentation.

• Facultative Anaerobe: Can use oxygen or not; both aerobic and anaerobic pathways.

• Aerotolerant Anaerobe: Tolerates oxygen but does not use it.

• Microaerophile: Requires low oxygen levels.

Summary Table: Oxygen Requirements

Type	Oxygen Use	Protective Mechanisms
Obligate Aerobe	Yes	Catalase, superoxide dismutase
Obligate Anaerobe	No	None or limited
Facultative Anaerobe	Yes/No	Catalase, superoxide dismutase
Aerotolerant Anaerobe	No	Superoxide dismutase
Microaerophile	Low	Limited catalase

Binary Fission vs. Mitosis

• Binary Fission: Prokaryotic cell division; produces genetically identical cells.

• Mitosis: Eukaryotic cell division; involves complex chromosome segregation.

Nutritional Requirements for Growth

• Macronutrients: C, N, P, S, O, H

• Micronutrients: Trace elements (Fe, Mg, Zn)

• Use: Building cellular structures, energy production.

Phases of Microbial Growth

• Lag Phase: Adaptation, no division.

• Log Phase: Exponential growth.

• Stationary Phase: Nutrient depletion, growth slows.

• Death Phase: Cell death exceeds division.

Trophic Classification of Microbes

• Phototrophs: Use light energy.

• Chemotrophs: Use chemical energy.

• Autotrophs: Use CO2 as carbon source.

• Heterotrophs: Use organic carbon.

Environmental Effects on Growth

• Temperature: Human pathogens are mesophiles (optimal 20-40°C).

• pH: Most pathogens prefer neutral pH.

• Osmotic Pressure: Isotonic environments preferred.

Bacterial Population Growth Calculations

• Exponential Growth: $N = N_0 \times 2^n$ where $N_0$ is initial cells, $n$ is generations.

Toxic Forms of Oxygen and Protective Enzymes

• Singlet Oxygen: Protected by carotenoids.

• Superoxide Radical: Protected by superoxide dismutase.

• Peroxide Anion: Protected by catalase, peroxidase.

• Hydroxyl Radical: No specific enzyme; minimized by antioxidants.

Biofilms and Quorum Sensing

• Biofilms: Communities of microbes attached to surfaces; resistant to antimicrobials.

• Quorum Sensing: Cell communication to coordinate behavior.

Culturing Bacteria

• Defined Media: Exact chemical composition known.

• Complex Media: Contains unknown components.

• Differential Media: Distinguishes between organisms.

• Selective Media: Favors growth of specific microbes.

Pure Culture Techniques

• Streak Plate: Isolates colonies.

• Pour Plate: Quantifies bacteria.

• CFU (Colony Forming Unit): Estimate of viable cells.

• Axenic Culture: Pure culture.

• Aseptic Technique: Prevents contamination.

Cell Counting Methods

• Turbidity Measurement: Uses spectrophotometer.

• Viable Count: Counts living cells.

Microbial Genetics

Ames Test and Auxotrophs

• Ames Test: Detects mutagenicity; does not identify carcinogenicity.

• Auxotroph: Mutant requiring additional nutrients.

Types of Mutations

• Silent: No effect on protein.

• Nonsense: Introduces stop codon.

• Missense: Changes amino acid.

• Frameshift: Alters reading frame.

Nucleic Acid Structure

• Nucleotide: 5-carbon sugar, phosphate, nitrogenous base.

• DNA: Double helix; stores genetic information.

• RNA: Single-stranded; types include mRNA, tRNA, rRNA.

Genetic Code and RNA Roles

• Genetic Code: Specifies amino acids.

• mRNA: Messenger; carries code.

• tRNA: Transfer; brings amino acids.

• rRNA: Ribosomal; forms ribosomes.

Replication, Transcription, Translation

• Replication: DNA copied; semiconservative.

• Transcription: DNA to RNA; requires RNA polymerase.

• Translation: RNA to protein; requires ribosomes, tRNA.

Mutagens and Effects

• Mutagen: Agent causing mutations.

• Effects: Can cause cancer, genetic disorders, or cell death.

Horizontal Gene Transfer

• Transformation: Uptake of naked DNA.

• Transduction: DNA transfer via bacteriophage (specialized/general).

• Conjugation: DNA transfer via pilus.

Chromosomes and Plasmids

• Prokaryotic Chromosomes: Circular, single.

• Eukaryotic Chromosomes: Linear, multiple.

• Plasmids: Small, circular DNA; F plasmid involved in conjugation.

Genetic Recombination

• Occurs: During horizontal gene transfer.

• Result: New genetic combinations.

Proofreading and Error Rates

• DNA Polymerase: Proofreads; low error rate.

• RNA Polymerase: No proofreading; higher error rate.

Operons: Inducible vs. Repressible

• Inducible Operon: Activated by substrate (e.g., lactose operon).

• Repressible Operon: Turned off by end product.

• Promoter: DNA sequence where RNA polymerase binds.

Lactose Operon

• Structure: Includes promoter, operator, structural genes.

• Function: Regulates lactose metabolism; ensures homeostasis.

Controlling Microbial Growth in the Environment

Ideal Antimicrobial Agents

• Characteristics: Effective, safe, stable, inexpensive.

Physical Methods of Microbial Control

• Categories: Heat, filtration, radiation.

• Mechanisms: Denaturation, removal, DNA damage.

• Examples: Autoclaving, membrane filtration, UV light.

Chemical Methods of Microbial Control

• Categories: Alcohols, halogens, oxidizing agents.

• Mechanisms: Protein denaturation, membrane disruption.

• Examples: Ethanol, bleach, hydrogen peroxide.

Microbial Death Rate and Time

• Death Rate: Rate at which microbes are killed.

• Death Time: Time required to kill all microbes.

• -static vs. -cidal: -static inhibits growth; -cidal kills microbes.

Inactivation of Protozoal Cysts and Bacterial Endospores

• Protozoal Cysts: Require high heat or chemicals.

• Bacterial Endospores: Require autoclaving or strong chemicals.

Biosafety Levels

• BSL-1: Minimal risk.

• BSL-2: Moderate risk.

• BSL-3: High risk; respiratory protection.

• BSL-4: Extreme risk; full containment.

Microbial Susceptibility to Antimicrobials

• Relative Susceptibility: Varies by species and structure.

Tests for Efficacy of Antiseptics and Disinfectants

• Methods: Use-dilution, disk-diffusion.

Key Definitions

• Antiseptic: Used on living tissue.

• Disinfectant: Used on surfaces.

• Filtration: Removal of microbes.

• Lyophilization: Freeze-drying.

• Desiccation: Drying.

• Ionizing Radiation: X-rays, gamma rays.

• Non-ionizing Radiation: UV light.

• Disinfect: Remove pathogens.

• Sanitize: Reduce microbes to safe levels.

• Degerm: Remove microbes from surface.

• Aseptic: Free from contamination.

• Axenic: Pure culture.

Controlling Microbial Growth in the Body: Antimicrobial Drugs

Types of Antimicrobial Agents

• Antifungals, Antibacterials, Anthelminthics, Antivirals, Antiprotozoals: Target specific pathogens.

• Fewest Agents: Antivirals; viruses use host machinery, making selective toxicity difficult.

Kirby-Bauer Susceptibility Test

• Purpose: Measures effectiveness of antibiotics.

• Method: Disk diffusion; zone of inhibition indicates susceptibility.

Mechanisms of Action and Selective Toxicity

• Mechanisms: Inhibit cell wall, protein synthesis, nucleic acid synthesis, metabolic pathways.

• Selectivity: Targets unique microbial features.

Specific Antimicrobial Classes

• Beta Lactam: Inhibits cell wall synthesis (e.g., penicillin).

• Sulfonamide: Inhibits folic acid synthesis.

• Antisense Nucleic Acids: Block translation by binding mRNA.

Antibiotic Resistance

• Development: Mutation, acquisition of resistance genes (lactamase, R-plasmids).

• Mechanisms: Enzyme production, efflux pumps, altered targets.

• Natural Selection: Resistant bacteria survive and proliferate.

Semi-Synthetic Drugs

• Advantage: Improved efficacy, reduced resistance.

Spectrum of Antibiotics

• Broad Spectrum: Effective against many species.

• Narrow Spectrum: Effective against specific species.

Recombinant DNA Technology (Optional)

Recombinant Vector Production

• Vector: DNA molecule used to carry gene insert.

• Process: Insert human gene into plasmid; use restriction enzymes and DNA ligase.

Reverse Transcriptase in Gene Production

• Function: Converts mRNA to cDNA for cloning.

Polymerase Chain Reaction (PCR)

• Purpose: Amplifies DNA.

• Enzyme: Taq polymerase from Thermus aquaticus.

Gel Electrophoresis

• Purpose: Separates DNA fragments by size.

Northern vs. Southern Blot

• Northern Blot: Detects RNA.

• Southern Blot: Detects DNA.

DNA Fingerprinting

• Technique: Uses restriction enzymes and gel electrophoresis.

• Uses: Identification, forensics.

Key Definitions

• Recombinant DNA: DNA from different sources.

• Xenotransplantation: Transplant from different species.

• Transgenic Organisms: Organisms with foreign genes.

• DNA Ligase: Joins DNA fragments.

• Restriction Enzyme: Cuts DNA at specific sites.

• cDNA: Complementary DNA.

• Mutagen: Causes mutations.

• Antisense RNA: Inhibits gene expression.

• DNA Polymerase: Synthesizes DNA.

• Splicing: Removal of introns.

• Plasmid: Circular DNA.

• DNA Probe: Detects specific DNA sequences.