Microbial Genetics

Introduction to Microbial Genomes

Microbial genetics explores the structure, function, and transmission of genetic material in microorganisms. Understanding these principles is essential for comprehending microbial physiology, evolution, and biotechnology applications.

• Genome: The complete set of genetic material in a cell or virus. In cells and DNA viruses, this is DNA; in RNA viruses, it is RNA.

• Bacterial genomes: Typically range from 1.5 to 6 million base pairs (bp).

Structure of Nucleic Acids

DNA Structure

DNA is a double-stranded molecule with specific structural features that enable its function as genetic material.

• Complementary base pairing: Adenine pairs with thymine, and guanine pairs with cytosine via hydrogen bonds.

• Antiparallel strands: The two DNA strands run in opposite directions (5' to 3' and 3' to 5').

• 5' end: Terminates at a phosphate group attached to the 5' carbon of deoxyribose.

• 3' end: Terminates with a hydroxyl group on the 3' carbon.

Organization of Prokaryotic and Eukaryotic Genomes

• Chromosomal DNA: Folded into loops (50,000–100,000 bp) stabilized by proteins and RNA.

• Histones: In archaea and eukaryotes, DNA wraps around positively charged histone proteins.

• Chromosome shape: Many prokaryotes have circular chromosomes; some have linear chromosomes.

• Plasmids: Small, circular DNA molecules (1%–20% the size of the chromosome) carrying non-essential genes, such as:

  • Fertility factors (F plasmids): Genes for conjugation.

  • Resistance factors (R plasmids): Genes for antimicrobial or heavy metal resistance.

  • Bacteriocin plasmids: Genes for toxins targeting similar bacteria.

  • Virulence plasmids: Genes for pathogenicity factors.

• Mitochondrial chromosomes: Circular DNA.

• Chloroplast chromosomes: Linear DNA.

Nucleotides and Their Functions

• Deoxyribonucleotides (e.g., dGTP): Building blocks of DNA, consisting of a base (guanine), deoxyribose sugar, and three phosphates. They also provide energy for DNA synthesis.

DNA Replication

Overview

DNA replication is a semiconservative process, ensuring genetic continuity during cell division.

• DNA helicase: Unzips DNA by breaking hydrogen bonds, forming a replication fork.

• Single-strand binding proteins: Stabilize separated DNA strands.

• DNA polymerase III: Main enzyme for synthesizing new DNA strands in bacteria.

Leading and Lagging Strand Synthesis

• Leading strand: Synthesized continuously toward the replication fork.

• Primase: Synthesizes a short RNA primer to initiate DNA synthesis.

• DNA polymerase III: Adds nucleotides to the 3' end, proofreads for errors.

• Lagging strand: Synthesized discontinuously away from the fork in Okazaki fragments.

• DNA polymerase I: Replaces RNA primers with DNA.

• DNA ligase: Seals nicks between Okazaki fragments.

Bidirectional replication: Two replication forks proceed in opposite directions.

Methylation: Addition of methyl groups (usually to adenine) for gene regulation and DNA protection.

Genotype and Phenotype

• Genotype: The complete set of genes (DNA sequences) in an organism.

• Phenotype: Observable characteristics resulting from gene expression, including structure, metabolism, and morphology.

Types of RNA and Their Functions

• RNA primer: Short RNA sequence required for DNA replication initiation.

• mRNA (messenger RNA): Carries genetic information from DNA to ribosomes.

• rRNA (ribosomal RNA): Combines with proteins to form ribosomes.

• tRNA (transfer RNA): Delivers amino acids to ribosomes during translation.

• Regulatory RNA: Modulates gene expression.

• Ribozymes: RNA molecules with enzymatic activity.

Transcription

Initiation

• RNA polymerase: Binds to promoter regions on DNA to start transcription.

• Sigma factor: Protein component that helps RNA polymerase recognize promoters.

Elongation

• RNA polymerase synthesizes RNA by linking ribonucleotides complementary to the DNA template strand.

• No primer is needed; only one DNA strand is transcribed.

• Transcription is slower than DNA replication.

Termination

• Self-termination: Occurs when RNA forms a hairpin structure at a GC-rich region followed by an A-rich region, causing RNA polymerase to dissociate.

• Rho-dependent termination: Rho protein binds to RNA and pushes RNA polymerase off the DNA.

Post-Transcriptional Modifications (Eukaryotes)

• Capping: Addition of a modified guanine nucleotide to the 5' end.

• Splicing: Removal of introns (non-coding regions); exons (coding regions) are joined.

• Transcription factors: Proteins that regulate the binding and activity of RNA polymerase.

The Genetic Code and Translation

The Genetic Code

• Codons: Triplets of mRNA nucleotides specifying amino acids.

• 61 codons code for amino acids; 3 are stop codons (UAA, UAG, UGA).

• AUG is the start codon (codes for methionine or formylmethionine in prokaryotes, mitochondria, and chloroplasts).

Translation Process

• In prokaryotes, translation can begin before transcription is complete (coupled transcription-translation).

• tRNA molecules (~75 nucleotides) have an anticodon complementary to mRNA codons.

• Ribosomes in mitochondria and chloroplasts resemble prokaryotic 70S ribosomes.

• Antibiotic example: Erythromycin binds to 23S rRNA, inhibiting protein synthesis.

Ribosome Sites

• A site: Entry point for tRNA carrying amino acids.

• P site: Holds tRNA with the growing polypeptide chain.

• E site: Exit site for discharged tRNAs.

Stages of Translation

• Initiation: Formation of the initiation complex (small and large ribosomal subunits, mRNA, initiator tRNA, and protein factors).

• Elongation: Sequential addition of amino acids, catalyzed by ribozymes and powered by GTP.

• Termination: Release factors recognize stop codons, and the polypeptide is released from the ribosome.

Gene Regulation: Operons

Operon Structure and Function

• Operon: A cluster of genes under the control of a single promoter and operator, often encoding proteins with related functions.

• Operator: DNA sequence that regulates access of RNA polymerase to the genes.

• Polycistronic mRNA: Single mRNA molecule encoding multiple polypeptides.

Types of Operons

• Inducible operons: Usually off; activated in response to specific substrates (e.g., lac operon for lactose metabolism).

• Repressible operons: Usually on; repressed when end products are abundant (e.g., trp operon for tryptophan synthesis).

Example: The Lac Operon

• Composed of a promoter, operator, and three structural genes for lactose transport and catabolism.

• Induced by:

  • Positive regulation via CAP (catabolite activator protein) and cAMP (increased when glucose is low).

  • Deactivation of the repressor protein.

Example: The Trp Operon

• Repressor is inactive by default; tryptophan activates the repressor, which then binds the operator to halt transcription.

Mutations and DNA Repair

Types of Mutations

• Gross mutations: Large-scale changes such as inversion, duplication, and transposition.

• Silent mutations: No change in amino acid sequence.

• Missense mutations: Change one amino acid; effects vary.

• Nonsense mutations: Introduce premature stop codons, usually resulting in nonfunctional proteins.

Mutagens

• Physical mutagens: Ionizing radiation (X-rays, gamma rays), nonionizing radiation (UV light).

• Chemical mutagens: Various chemicals that alter DNA structure.

DNA Repair Mechanisms

• Direct repair: Corrects errors in one DNA strand.

• Base-excision repair: Removes and replaces incorrect bases.

• Light repair: Fixes UV-induced pyrimidine dimers.

• Single-strand repair: Removes and replaces damaged DNA sections.

Detection and Selection of Mutants

• Positive selection: Identifies mutants by eliminating wild-type phenotypes.

• Auxotroph: Mutant organism requiring different nutrients than the wild type.

• Ames test: Rapid, inexpensive screening for mutagens using liver extract to simulate metabolic activation.

Horizontal Gene Transfer in Prokaryotes

Overview

Horizontal (lateral) gene transfer allows genetic exchange between different cells, sometimes even across species, contributing to genetic diversity.

• Occurs in less than 1% of prokaryotic populations.

Mechanisms of Horizontal Gene Transfer

Summary Table: Key Terms and Concepts

Key Equations

• Base pairing:

• Directionality of DNA synthesis:

Additional info:

• Some context on the Ames test and DNA repair mechanisms was expanded for clarity.

• Tables were inferred and constructed to summarize horizontal gene transfer and key terms.