Microbial Genetics: DNA Replication, Gene Expression, Regulation, and Mutation

Microbial Genetics

Introduction to Microbial Genetics

Microbial genetics explores how microorganisms store, copy, express, regulate, and exchange genetic information. Understanding these processes is fundamental to microbiology, as they underpin microbial function, adaptation, and evolution.

• Genetics: The study of heredity and variation in organisms.

• Genome: The complete genetic information of an organism.

• Gene: A segment of DNA coding for a functional product, usually a protein or sometimes RNA.

• Chromosome: A large DNA molecule containing essential genes; bacteria typically have one circular chromosome.

• Plasmid: Small, circular DNA molecules that replicate independently and carry accessory genes (e.g., antibiotic resistance, virulence factors).

Example: Plasmids may confer antibiotic resistance, allowing bacteria to survive in the presence of antibiotics.

DNA Structure

DNA is the hereditary material in most organisms, structured as a double helix composed of nucleotides.

• Nucleotide: Contains a phosphate group, deoxyribose sugar, and a nitrogenous base.

• Bases: Purines (Adenine, Guanine) and Pyrimidines (Cytosine, Thymine).

• Base Pairing: Adenine pairs with Thymine (A-T), Guanine pairs with Cytosine (G-C).

• Complementary Strands: Each strand can be predicted from the other due to specific base pairing.

• Antiparallel Orientation: One strand runs 5'→3', the other 3'→5'.

Example: If one DNA strand reads 5'-ATGC-3', the complementary strand reads 3'-TACG-5'.

DNA Replication

DNA replication is the process by which a cell copies its DNA before cell division (binary fission). It is semiconservative, meaning each daughter DNA molecule contains one parental and one newly synthesized strand.

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

• Replication Fork: The site where DNA strands separate and replication occurs.

• Enzymes Involved:

  • Helicase: Unwinds DNA.

  • Primase: Synthesizes RNA primer.

  • DNA Polymerase III: Synthesizes new DNA strand (5'→3').

  • DNA Polymerase I: Removes RNA primers and fills gaps.

  • DNA Ligase: Seals fragments, making the strand continuous.

• Leading Strand: Synthesized continuously in the 5'→3' direction.

• Lagging Strand: Synthesized discontinuously as Okazaki fragments, each requiring an RNA primer.

Replication Steps:

1. DNA strands separate.

2. Replication fork forms.

3. RNA primer attaches.

4. DNA Polymerase synthesizes DNA.

5. Leading strand is continuous; lagging strand is discontinuous.

6. RNA primers removed; DNA Ligase seals fragments.

Equation:

$\text{DNA}_{\text{parent}} \rightarrow \text{DNA}_{\text{daughter}}$

Example: DNA replication must occur before binary fission to ensure each daughter cell receives a complete genome.

Gene Expression: Transcription & Translation

Gene expression involves two main processes: transcription (DNA to RNA) and translation (RNA to protein).

Transcription

• Definition: Synthesis of RNA from a DNA template.

• RNA Types: mRNA (messenger), tRNA (transfer), rRNA (ribosomal).

• Steps:

  1. RNA polymerase binds promoter.

  2. DNA unwinds.

  3. RNA synthesized 5'→3'.

  4. Terminator reached; RNA released.

Equation:

$\text{DNA} \xrightarrow{\text{Transcription}} \text{RNA}$

Eukaryotic RNA Processing

• Exons: Expressed sequences; code for protein.

• Introns: Non-coding sequences; removed from pre-mRNA.

• Ribozymes: RNA molecules that act as enzymes to remove introns.

• Occurs only in eukaryotes.

Translation

• Definition: Synthesis of protein from mRNA.

• Players: mRNA, tRNA, rRNA.

• Codons: Three-base sequences on mRNA specifying amino acids.

• Start Codon: AUG (methionine).

• Stop Codons: UAA, UAG, UGA.

• tRNA: Contains anticodon complementary to mRNA codon.

• Ribosome: Composed of small and large subunits; catalyzes peptide bond formation.

• Sites: A site (incoming tRNA), P site (growing peptide chain).

Equation:

$\text{mRNA} \xrightarrow{\text{Translation}} \text{Protein}$

Example: The codon AUG codes for methionine and signals the start of translation.

Prokaryotes vs Eukaryotes

• Prokaryotes: Transcription and translation occur simultaneously in the cytoplasm.

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

Gene Regulation

Operon Structure

An operon is a cluster of genes under the control of a single promoter and operator, allowing coordinated regulation.

• Promoter: Site where RNA polymerase binds.

• Operator: Control switch; repressor binds here.

• Structural Genes: Genes being transcribed.

• Regulatory Gene: Produces repressor protein.

Types of Operons

• Inducible Operon: Default OFF; turned ON by inducer (e.g., lac operon).

• Repressible Operon: Default ON; turned OFF by corepressor (e.g., trp operon).

• Constitutive Enzymes: Produced continuously at a fixed rate; always ON.

Inducible Operon Example: lac Operon

• Regulatory gene: Produces active repressor.

• In absence of lactose: Repressor binds operator, blocking transcription.

• In presence of lactose: Lactose converted to allolactose (inducer), which inactivates repressor, allowing transcription.

• Structural genes: lacZ (β-galactosidase), lacY (permease), lacA (transacetylase).

Repressible Operon Example: trp Operon

• Regulatory gene: Produces inactive repressor.

• Low tryptophan: Repressor inactive; transcription occurs.

• High tryptophan: Tryptophan acts as corepressor, activates repressor, which binds operator and stops transcription.

Diauxic Growth

• Glucose: Preferred carbon source; used first.

• Lag phase: Cells synthesize enzymes for lactose metabolism.

• After lag: Lactose metabolized.

Example: Bacteria exhibit a lag when switching from glucose to lactose due to enzyme synthesis.

Mutations & DNA Repair

Mutations

A mutation is a change in the genetic material (DNA). Mutations can be neutral, beneficial, or harmful. Mutagens are agents that cause mutations, such as chemicals or radiation.

• Spontaneous Mutations: Occur naturally during DNA replication.

• Genotype: Genetic information.

• Phenotype: Expression of genes.

• Vertical Gene Transfer: Parent to daughter cell (binary fission).

• Horizontal Gene Transfer: Between cells of the same generation (recombination).

Types of Mutations

• Point Mutation (Base Substitution): One nucleotide replaced.

  • Missense Mutation: Changes one amino acid.

  • Nonsense Mutation: Creates a stop codon; protein synthesis stops early.

• Frameshift Mutation: Insertion or deletion of nucleotides shifts reading frame; usually has greater effect than point mutation.

Mutagens

• Ionizing Radiation: X-rays, gamma rays; produce ions, damage DNA and sugar-phosphate backbone.

• Non-Ionizing Radiation: Ultraviolet (UV); forms thymine dimers.

DNA Repair Mechanisms

• Proofreading: DNA polymerase checks and corrects errors during replication.

• Nucleotide Excision Repair: Damaged DNA removed; DNA polymerase fills gap; DNA ligase seals strand.

• Mismatch Repair: Corrects errors missed during replication.

Repair Sequence:

1. Damage recognized (e.g., thymine dimers).

2. Damaged DNA removed.

3. DNA polymerase fills gap.

4. DNA ligase seals strand.

DNA Surveillance

• p53 Gene: Located on chromosome 17; monitors DNA integrity and initiates repair.

• Repair efficiency: Higher in young cells; decreases with age.

Mutation Detection: Selection & Ames Test

• Positive (Direct) Selection: Mutant cells grow or appear different (e.g., antibiotic resistance).

• Negative (Indirect) Selection: Mutants identified by inability to grow (e.g., replica plating).

• Replica Plating: Transfer colonies to media with/without specific nutrients to identify mutants.

• Ames Test: Detects mutagenic chemicals using histidine-dependent Salmonella; growth on histidine-free plate indicates reverse mutation.

Summary Table: Operon Types

Operon Type	Default State	Regulation Mechanism	Example
Inducible	OFF	Inducer inactivates repressor	lac operon
Repressible	ON	Corepressor activates repressor	trp operon
Constitutive	ON	Always expressed	Housekeeping genes

Key Vocabulary

• Genetics

• Genome

• Gene

• Chromosome

• Plasmid

• DNA

• Nucleotide

• Purine

• Pyrimidine

• Complementary

• Antiparallel

• Semiconservative replication

• Replication fork

• Leading strand

• Lagging strand

• RNA primer

• Okazaki fragment

• DNA Polymerase

• DNA Ligase

• Mutation

• Mutagen

• Spontaneous mutation

• Point mutation

• Missense

• Nonsense

• Frameshift

• Insertion

• Deletion

• Ionizing radiation

• Non-ionizing radiation

• Thymine dimer

• DNA repair

• p53

• Positive selection

• Negative selection

• Replica plating

• Histidine-dependent

• Ames test

• Transcription

• Translation

• Promoter

• Terminator

• mRNA

• tRNA

• rRNA

• Codon

• Anticodon

• Start codon

• Stop codon

• Ribosome

• Small subunit

• Large subunit

• A site

• P site

• Exons

• Introns

• Ribozymes

• Peptide bond

Additional info: The Meselson-Stahl experiment, which demonstrated semiconservative replication, is considered supplemental and not emphasized in this course.