Microbial Metabolism

Enzymes and Their Function

Enzymes are biological catalysts that speed up chemical reactions in cells by lowering the activation energy required. They are essential for metabolic processes.

• Enzyme: A protein (sometimes RNA) that catalyzes a specific biochemical reaction.

• Substrate: The specific reactant that an enzyme acts upon.

• Cofactor: A non-protein component (such as a metal ion or coenzyme) required for enzyme activity.

• Mechanism: Enzymes bind substrates at the active site, forming an enzyme-substrate complex, and convert substrates into products.

• Specificity: Enzymes are highly specific for their substrates.

• Example: Amylase catalyzes the breakdown of starch into sugars.

Oxidation-Reduction (Redox) Reactions

Redox reactions involve the transfer of electrons between molecules, fundamental to energy production in cells.

• Oxidation: Loss of electrons (or hydrogen atoms).

• Reduction: Gain of electrons (or hydrogen atoms).

• Electron Carriers: Molecules such as NAD+, NADP+, and FAD that shuttle electrons during metabolic reactions.

• Reducing Power: The ability of molecules like NADH and FADH2 to donate electrons in metabolic pathways.

• Example: NAD+ + 2H → NADH + H+

Metabolism: Anabolism and Catabolism

Metabolism encompasses all chemical reactions in a cell, divided into two main types:

• Anabolism: Biosynthetic reactions that build complex molecules from simpler ones (requires energy).

• Catabolism: Degradative reactions that break down complex molecules into simpler ones (releases energy).

• Example: Glycolysis is a catabolic pathway; protein synthesis is anabolic.

Respiration and Fermentation

Microorganisms generate energy through various metabolic pathways:

• Aerobic Respiration: Uses oxygen as the final electron acceptor; yields the most ATP.

• Anaerobic Respiration: Uses inorganic molecules other than oxygen (e.g., nitrate, sulfate) as final electron acceptors.

• Fermentation: Organic molecules (e.g., pyruvate) serve as final electron acceptors; occurs without an electron transport chain.

• Final Electron Acceptors:

  • Aerobic: O2

  • Anaerobic: Nitrate (NO3-), sulfate (SO42-), etc.

  • Fermentation: Organic molecules (e.g., pyruvate, acetaldehyde)

• ATP Generation:

  • Substrate-level phosphorylation: Direct transfer of phosphate to ADP.

  • Oxidative phosphorylation: ATP synthesis via electron transport chain and chemiosmosis.

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

Major Metabolic Pathways and ATP Yield

Microbial cells use several key pathways to extract energy from nutrients:

• Glycolysis: Converts glucose to pyruvate; produces 2 ATP (substrate-level), 2 NADH.

• Transition (Decarboxylation) Step: Pyruvate to acetyl-CoA; produces 2 NADH per glucose.

• Krebs (TCA) Cycle: Acetyl-CoA oxidized to CO2; produces 2 ATP (substrate-level), 6 NADH, 2 FADH2 per glucose.

• Electron Transport Chain (ETC): NADH and FADH2 donate electrons; O2 (or other acceptor) is reduced; ATP is made by oxidative phosphorylation.

Additional info: The actual ATP yield may vary depending on the organism and conditions.

Fermentation Pathways

Fermentation allows ATP production in the absence of oxygen or an electron transport chain.

• Pyruvic Acid (Pyruvate): End product of glycolysis; reduced to various fermentation products.

• Lactic Acid Fermentation: Pyruvate reduced to lactic acid (e.g., in Lactobacillus).

• Alcoholic Fermentation: Pyruvate converted to ethanol and CO2 (e.g., in yeast).

Lipid and Protein Catabolism

Microbes can use lipids and proteins as energy sources when carbohydrates are unavailable.

• Lipids: Broken down by lipases into glycerol and fatty acids; fatty acids enter β-oxidation to form acetyl-CoA.

• Proteins: Degraded by proteases into amino acids; amino acids are deaminated and enter glycolysis or the TCA cycle.

Microbial Genetics

Genetic Material: Chromosomes, Plasmids, and Genes

Genetic information in microbes is organized in chromosomes and plasmids.

• Chromosome: Main DNA molecule containing essential genes.

• Plasmid: Small, circular DNA molecule; often carries non-essential, advantageous genes (e.g., antibiotic resistance).

• Gene: DNA segment encoding a functional product (protein or RNA).

• Genome: Complete set of genetic material in a cell.

DNA and RNA Structure

DNA and RNA are nucleic acids with distinct structures and functions.

• DNA: Double-stranded helix; deoxyribose sugar; bases: adenine (A), thymine (T), cytosine (C), guanine (G).

• RNA: Single-stranded; ribose sugar; bases: adenine (A), uracil (U), cytosine (C), guanine (G).

• Base-pairing rules: A-T (DNA), A-U (RNA), C-G.

Central Dogma: Replication, Transcription, Translation

Genetic information flows from DNA to RNA to protein.

• Replication: DNA is copied semi-conservatively; each new DNA has one old and one new strand.

• Transcription: DNA is used as a template to synthesize RNA (mRNA, tRNA, rRNA).

• Translation: mRNA is decoded by ribosomes to synthesize proteins.

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

• Anticodon: Three-nucleotide tRNA sequence complementary to mRNA codon.

• DNA Polymerase: Synthesizes DNA from a DNA template.

• RNA Polymerase: Synthesizes RNA from a DNA template.

Gene Transfer Mechanisms

Bacteria can exchange genetic material through horizontal and vertical gene transfer.

• Horizontal Gene Transfer (HGT):

  • Transformation: Uptake of naked DNA from the environment; requires competence; DNase sensitive.

  • Conjugation: Direct transfer via pilus; F+, Hfr, F- status refers to presence and integration of F plasmid.

  • Transduction: Transfer via bacteriophage (virus).

• Vertical Gene Transfer: Transmission of genetic material from parent to offspring during reproduction (binary fission, mitosis).

Mutations and Mutagenesis

Mutations are heritable changes in DNA sequence.

• Base Substitution: One base is replaced by another (missense, nonsense, silent mutations).

• Addition/Deletion: Insertion or removal of bases; may cause frameshift mutations.

• Mutagens: Agents that increase mutation rate (chemicals, X-rays, UV, transposable elements).

• Transposons: DNA segments that can move within the genome.

Selection Methods

• Direct Selection: Only mutants grow (e.g., antibiotic resistance).

• Indirect Selection: Mutants identified by replica plating (e.g., auxotrophs).

Genetic Applications and Biotechnology

Recombinant DNA Technology

Biotechnology uses genetic engineering to manipulate DNA for research, medicine, and industry.

• Recombinant DNA: DNA molecules formed by combining DNA from different sources.

• Genetic Recombination (Cloning): Inserting a gene of interest into a vector to produce multiple copies.

• Restriction Endonucleases (Enzymes): Cut DNA at specific sequences.

• Ligase: Joins DNA fragments together.

• Cloning Vector: DNA molecule (e.g., plasmid) used to carry foreign DNA into a host cell.

• Selectable Gene Markers: Genes (e.g., antibiotic resistance) used to identify cells with recombinant DNA.

• Colony Blotting: Technique to identify colonies with a specific DNA sequence.

Probes, Primers, and cDNA

• Probe: Labeled DNA or RNA sequence used to detect complementary sequences.

• Primer: Short DNA sequence that initiates DNA synthesis in PCR.

• cDNA (complementary DNA): DNA synthesized from mRNA by reverse transcriptase.

• Reverse Transcriptase: Enzyme that synthesizes DNA from an RNA template.

Polymerase Chain Reaction (PCR)

PCR is a technique to amplify specific DNA sequences rapidly and efficiently.

• Advantages: High sensitivity, specificity, and speed; requires only small amounts of DNA.

• Steps: Denaturation, annealing, extension.

Dideoxy Chain Termination (Sanger Sequencing)

• Method: DNA synthesis is terminated by incorporation of dideoxynucleotides, allowing determination of DNA sequence.

Summary Table: Key Concepts

Key Equations

• ATP yield from NADH and FADH2 in ETC:

• General redox reaction: