Bacterial Genetics

Bacterial Genetics

Major Mechanisms of Gene Transfer in Bacteria

  • Transduction

  • Conjugation

  • Transformation

Three Possible Fates of Incoming DNA

After entry, incoming DNA has three possible fates:

  1. Degradation by nucleases or restriction enzymes.

  2. Survival as an independent replicon (must be circular with origin of replication).

  3. Integration via homologous recombination into the host chromosome.

Oswald Avery’s Experiment (Proof DNA Is Genetic Material)

  • Used Streptococcus pneumoniae:

    • Smooth strain → virulent (has capsule).

    • Rough strain → nonvirulent.

  • Extracted DNA from smooth strain.

  • Added purified DNA to rough strain.

  • Rough bacteria became smooth and virulent.

  • Injecting transformed bacteria into mice killed them.

Conclusion: DNA alone carries genetic information.

Transduction

Two Types of Transduction

1⃣ Generalized Transduction

  • Random bacterial DNA fragments packaged.

  • Any gene may be transferred.

  • Example: P1 phage

2⃣ Specialized Transduction

  • Only genes near phage integration site transferred.

  • Due to incorrect excision of prophage.

  • Example: Lambda phage (attλ site)

Conjugation

  • Figures Reference: 28.12 and 28.14

  • Bacterial Conjugation:

    • Mechanism where genetic material is transferred via direct contact between bacteria, often through a pilus.

    • Integration of F-plasmid: Figures 28.16 and 28.17 show how F-plasmids can be integrated into chromosomal DNA.

    • Transfer of Chromosomal Genes: Figures 28.18 and 28.19 detail the role of Hfr strains in facilitating chromosomal gene transfer.

Transformation

  • Uptake of naked DNA from the environment.

  • DNA must recombine with the chromosome or be a replicon to survive

  • Key Concept: Gene transfer where cells can uptake free DNA from their environment.

  • Recombination: Allows for survival and incorporation of transformed DNA,

  • Competence Pheromones: signaling molecules that induce competence in bacteria.

Natural Competence

Competent

A competent cell is capable of taking up DNA from its environment

Competence Pheromones

  • Short secreted peptides.

  • Accumulate at high cell density.

  • Bind receptors on nearby cells.

Induce genes for DNA uptake

This ensures DNA uptake occurs when many related cells are nearby.

Feature

Natural Competence

Artificial Competence

Trigger

Pheromones at high cell density

Lab treatment

Mechanism

Protein-mediated uptake

Cell wall damage

DNA Entry

Single-stranded DNA enters

Double-stranded DNA enters

Survival

Controlled physiological process

Many cells die

Differences in Ca2+ Heat Shock vs Electroporation:

Feature

Chemical (Ca²⁺/Heat Shock)

Electroporation

Method

Calcium weakens membrane

High-voltage pulse

DNA Form

dsDNA enters

dsDNA enters

Cell Damage

Moderate

Often severe

Efficiency

Moderate

High

Typical Use

E. coli

Yeast, difficult strains

Applications of Bacterial Transformation

  • Molecular Biology Applications:

    • Cloning to make multiple copies of DNA.

    • Expressing large amounts of proteins and enzymes.

    • Generating genomic and cDNA libraries.

    • Conducting DNA linkage studies.

How Vibrio cholerae Kills and Steals DNA

  • Uses Type VI Secretion System (T6SS).

  • Injects toxic proteins into neighboring bacteria.

  • Kills them.

  • Released DNA is taken up.

  • DNA uptake machinery induced by:

    • High cell density

    • Chitin fragments

This is aggressive horizontal gene transfer.

Key Steps in Bacterial Transformation

  1. Competent Cell Preparation

    • Chemically Competent Cells:

      • Maintained in calcium chloride (CaCl2) to increase permeability of the cell envelope.

      • Heat shock opens pores temporarily for DNA uptake.

    • Electrocompetent Cells:

      • Prepared for electroporation, washed to remove interfering salts and resuspended in 10% glycerol for storage.

  2. Transformation Step

    • Chemical/Heat Shock Method:

      • Utilizes Ca2+ ions and temperature shifts for DNA entry into the bacterial cytoplasm during heat shock (typically 42°C for 30-120 seconds).

    • Electroporation Method:

      • Uses electrical current to induce small pores in the cellular envelope, allowing plasmid entry.

      • Procedure involves mixing plasmid with electrocompetent cells subjected to an electrical shock.

  3. Cell Recovery Period:

    • Involves adding antibiotic-free media for approximately 1 hour to facilitate expression of antibiotic resistance genes, support cell division, and improve viability and cloning efficiency.

  4. Cell Plating:

    • After recovery, cells are plated on selective/differential media to obtain distinct colonies.

    • Important to account for expiration of antibiotic plates and pre-warm plates.

    • Aim for adequate colony spacing to avoid satellite colonies from antibiotic breakdown around large colonies.

Experimental Design: Transformation Controls

Positive Control

  • Cells transformed with known working plasmid.

  • Confirms cells are competent and antibiotic works.

Negative Control

  • Cells with no DNA.

  • Should not grow on selective media.

  • Confirms antibiotic selection is functioning.

Controls ensure:

  • DNA uptake occurred.

  • Selection is valid.

  • No contamination.

Learning Objectives

  • Understand the three mechanisms of bacterial gene transfer.

  • Identify possible fates of incoming DNA fragments during gene transfer.

  • Analyze Oswald Avery’s experiment demonstrating DNA as the genetic material.

  • Define "competent" and discuss competence pheromones.

  • Compare natural competence to artificially induced competence mechanisms.

  • Explain how Vibrio cholerae kill bacteria and acquire DNA.

  • Describe the two types of transduction.

  • Detail key steps in bacterial transformation for molecular biology applications.

  • Evaluate differences between chemical transformation and electroporation methods.

  • Discuss importance of positive and negative controls in transformation experiments.