Microbial Genetics: Structure, Function, and Variation

Genetics Fundamentals & DNA Structure

Core Definitions

Genetics is the study of how genetic information is stored, expressed, and transmitted in living organisms. In microbiology, understanding the structure and function of genetic material is essential for grasping microbial diversity and adaptation.

• Genetics: The study of genes, their functions, and inheritance.

• Chromosomes: DNA-containing structures; bacteria typically have a single, circular chromosome.

• Genes: DNA segments encoding functional products, usually proteins.

• Genome: The complete set of genetic material in a cell.

• Genotype: The genetic constitution of an organism.

• Phenotype: Observable traits resulting from gene expression.

• Short Tandem Repeats (STRs): Repetitive, noncoding DNA sequences.

Vertical Gene Transfer

• Genetic information is passed from parent to offspring during cell division.

• Binary Fission: Bacterial cells replicate their chromosome before dividing, ensuring genetic continuity.

• Fidelity: DNA polymerase proofreading ensures high accuracy, producing genetically identical clones.

DNA Structure

• Chemical Composition: DNA consists of a sugar-phosphate backbone and nitrogenous bases.

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

• Complementary Base Pairing: Adenine pairs with Thymine (A-T), and Cytosine pairs with Guanine (C-G) via hydrogen bonds.

Example: In Escherichia coli, the genome is a single circular DNA molecule containing thousands of genes.

The Central Dogma & Replication Machinery

The Central Dogma

The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to protein, forming the basis for gene expression.

• Genetic Code: The set of rules by which nucleotide sequences are translated into amino acid sequences.

• Pathway: DNA $\rightarrow$ RNA $\rightarrow$ Protein

Replication Machinery

• Topoisomerase & Gyrase: Enzymes that relax supercoiled DNA before replication.

• Helicase: Unwinds the double helix, separating the two DNA strands.

• DNA Polymerase: Synthesizes new DNA strands in the 5' $\rightarrow$ 3' direction.

• DNA Ligase: Joins Okazaki fragments on the lagging strand.

Replication Strands & Accuracy

• Leading Strand: Synthesized continuously toward the replication fork.

• Lagging Strand: Synthesized discontinuously as Okazaki fragments, later joined by DNA ligase.

• Bidirectional Replication: In bacteria, replication proceeds in both directions from the origin, increasing efficiency.

• Proofreading: DNA polymerase corrects errors, ensuring high fidelity.

Example: During E. coli replication, the entire genome is copied in about 40 minutes with remarkable accuracy.

RNA, Transcription, & Translation

RNA Structure & Types

RNA is a single-stranded nucleic acid with ribose sugar and uracil instead of thymine. It plays multiple roles in gene expression.

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

• tRNA (Transfer RNA): Brings amino acids to the ribosome during protein synthesis.

• rRNA (Ribosomal RNA): Forms the core of ribosome structure and catalyzes protein synthesis.

Translation & The Codon Language

• Codons: Triplets of nucleotides in mRNA specifying amino acids.

• Degeneracy: Multiple codons can code for the same amino acid (61 sense codons for 20 amino acids).

• Start Codon: AUG (codes for methionine).

• Stop Codons: UAA, UAG, UGA signal termination of translation.

• Bacterial Advantage: In prokaryotes, translation can begin before transcription is complete due to the absence of a nuclear membrane.

Eukaryotic RNA Processing

• Compartmentalization: Transcription occurs in the nucleus; translation in the cytoplasm.

• Exons: Coding regions of DNA.

• Introns: Noncoding regions removed from pre-mRNA.

• snRNPs: Small nuclear ribonucleoproteins that splice exons together.

Example: In eukaryotes, the gene for hemoglobin contains both exons and introns; introns are removed before translation.

Bacterial Gene Regulation (Operons & Epigenetics)

Control Mechanics

Bacteria regulate gene expression to adapt to environmental changes, using operons and other mechanisms.

• Constitutive Genes: Expressed continuously at a fixed rate.

• Repression: Repressor proteins block transcription.

• Induction: Inducers activate transcription.

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

• Operator: DNA segment controlling access of RNA polymerase to structural genes.

• Inducible Operon (lac operon): Activated in the presence of lactose.

• Repressible Operon (trp operon): Turned off when tryptophan is abundant.

Complex Regulation & Epigenetics

• Catabolite Repression: Glucose presence inhibits the use of other sugars; absence of glucose increases cAMP, which binds to CAP to enhance transcription of alternative sugar operons.

• Epigenetics (Methylation): Chemical modifications (e.g., methylation) can silence genes without altering the DNA sequence and may be inherited.

Example: The lac operon in E. coli is a classic model for inducible gene regulation.

Mutations & Genetic Selection

Mutation Categories

Mutations are permanent changes in the DNA sequence, which can affect gene function and phenotype.

• Base Substitution: Replacement of one nucleotide with another.

• Missense Mutation: Alters the amino acid sequence of a protein.

• Nonsense Mutation: Introduces a premature stop codon, truncating the protein.

• Frameshift Mutation: Insertion or deletion shifts the reading frame, altering downstream amino acids.

Mutagens

• Chemical Mutagens: Nitrous acid causes A to pair with C; nucleotide analogs cause mispairing.

• Radiation Mutagens: Ionizing radiation breaks DNA; UV light causes thymine dimers.

• DNA Repair: Photolyases and excision repair enzymes correct DNA damage.

Mutation Rates & Identification

• Spontaneous Mutation Rate: Approximately 1 in $10^9$ base pairs or 1 in $10^6$ genes per replication.

• Mutagen-Induced Rate: Increases to $10^{-5}$ to $10^{-3}$ per gene.

• Positive (Direct) Selection: Identifies mutants by their ability to grow or appear differently.

• Negative (Indirect) Selection: Identifies mutants by their inability to grow or perform a function.

• Auxotroph: A mutant requiring a nutrient not needed by the parent.

• Ames Test: Assesses mutagenicity by measuring the reversion rate of mutant bacteria exposed to chemicals.

Example: The Ames test is widely used to screen chemicals for potential carcinogenicity.

Recombination, Plasmids, & Horizontal Gene Transfer

Recombination & Vectors

Genetic recombination increases diversity by exchanging DNA between molecules. Plasmids and other vectors facilitate gene transfer in bacteria.

• Genetic Recombination: Exchange of DNA segments between molecules, creating new gene combinations.

• Crossing Over: Chromosomes break and rejoin, incorporating foreign DNA.

• Plasmids: Small, self-replicating circular DNA molecules, often carrying beneficial genes.

• Conjugative Plasmids: Encode sex pili for plasmid transfer between cells.

• Resistance Factors (R Factors): Plasmids carrying antibiotic resistance genes, contributing to drug resistance.

Transposons & Mechanisms

• Transposons: DNA segments that move within the genome, containing insertion sequences and the enzyme transposase.

• Transformation: Uptake of naked DNA from the environment by a cell.

• Griffith's Experiment (1928): Demonstrated transformation by showing that non-virulent bacteria could acquire virulence from heat-killed virulent strains.

Mapping & Transduction

• Chromosome Mapping via Conjugation: Hfr strains transfer genes in a linear sequence; mapping is measured in minutes, indicating gene order and distance.

• Transduction: Bacteriophages transfer bacterial genes between cells.

• Generalized Transduction: Any gene can be transferred during the lytic cycle.

• Specialized Transduction: Only specific genes near the prophage site are transferred.

Example: R factor plasmids are a major concern in hospital-acquired infections due to their role in spreading antibiotic resistance.

Evolution

Summary

Genetic diversity in microbial populations arises from mutation and recombination. Natural selection acts on this variation, favoring traits that enhance survival and reproduction.

• Mutation: Introduces new genetic variants.

• Recombination: Shuffles existing genes to create new combinations.

• Natural Selection: Favors advantageous traits, shaping microbial evolution.

Example: The rapid emergence of antibiotic-resistant bacteria is a direct result of mutation, gene transfer, and selection pressure from antibiotic use.