Lecture_15__Horizontal_Gene_Transfer

Evolution and Genetic Transfer

• Evolution: Acts at the level of individual genes.

• Transformation: Uptake of naked DNA from the environment and incorporation into the genome.

  -Example: Streptococcus pneumoniae can take up antibiotic resistance genes from dead bacteria.

• Transduction: Transfer of DNA mediated by bacteriophage infection.

  -Some bacteria gain toxin-producing genes through phage infection (e.g., diphtheria toxin).

• Transposition: "Hopping" of self-mobilizing elements of DNA within the genome or between bacteria.

• Conjugation: Cell-contact dependent transfer of DNA between bacteria.

Learning Objectives

• Understand Horizontal Gene Transfer including:

  • Plasmids

  • Conjugation

  • Transformation

• Recognize that frequencies of gene transfers can change independently from the organisms involved.

• Acknowledge the enzymatic activity involved in moving DNA from one spot in the genome to another.

Plasmids

• Cryptic Plasmid: Small plasmid (2000 nt) widely found in gut microbes.

  • Incompatibility group protein: Prevents other plasmids from entering the bacterium.

  • Antibiotic resistance markers: Determine what bacteria can uptake the plasmid including the gene bla which codes for β-lactamase (a key protein that breaks down antibiotics).

• Origin of replication (ori): Point where plasmid replication begins.

• Multiple Cloning Site (MCS): Contains multiple restriction sites for restriction enzymes to cut DNA.

  • Restriction enzymes: Cut DNA at specific regions.

  • Methylation: Prevents cleavage by methylating nucleotides.

  • lacZ gene: A reporter gene used in blue-white screening; blue indicates no DNA insertion.

• Megaplasmid: Very large plasmids (up to 1 Mb) that border on being second chromosomes.

Properties of Plasmids

• Some plasmids are very mobile and can be parasitic, offering no evolutionary advantage to the host.

• Size range: 3-100+ kb, typically containing only genes for replication and horizontal transfer.

• Small plasmids often carry only essential replication and transfer genes.

• Larger plasmids can include genes for antibiotic resistance, virulence, or metabolism.

Artificial Cloning Vectors

• Range from 3-10 kb with characteristics:

  • Increased copy number based on naturally occurring plasmids.

  • Can have a broad or narrow host range, affected by compatibility with incompatibility group proteins.

• Natural plasmids are often much larger and may contain unique abilities to cleave unwanted foreign DNA.

Examples of Plasmids in Action

• EcoR1: Restriction enzyme from E.Coli; can insert DNA into plasmids.

• LacZ Screening: Indicates if DNA was inserted by observing color change in the presence of X-Gal.

Natural Plasmids and Replication

• Note that natural plasmids are widespread and enhance bacterial fitness through easy DNA transfer.

• Conjugation bridge formation: Involves the formation of pilus and transfer of genetic material.

F Plasmid and Its Role in Conjugation

• F Plasmid: Carries genes necessary for conjugation and can form composite plasmids with various traits.

• Homologous Recombination: Involves crossing over of homologous sequences and formation of Holliday junctions.

  • Site-specific recombination involving integrase enzymes.

Transformation Mechanism

• Transformation: Uptake of naked DNA by bacterial cells was first discovered in Griffith's experiment.

  • Techniques include Calcium Chloride Heat Shock and Electroporation.

• Natural competence: Some microbes can naturally take up DNA and integrate it into their genomes.

  • Bacteria like S. pneumoniae and Neisseria gonorrheae demonstrate this ability.

Common Themes in Transformation

• Involvement of DNA binding proteins, nuclease activity, and integration of single-strand DNA.

• Many bacteria reside in biofilms, with DNA being a main component of the biofilm matrix.

Detailed Conjugation Process

• Conjugation requires cell-cell contact, often mediated by a sex pilus that enables the transfer of plasmids.

• Hfr strains: Strains with F-plasmid DNA integrated into their chromosomes facilitate transfer to F-strains.

  • The mapping of E. coli's chromosome relies on the characteristics of Hfr mating.

Conjugation Experiments

• Early experiments by Josh and Ester Lederberg and later by Davis in 1950 established methodologies to test DNA transfer.

• U-tube experiments were conducted to exclude transformation, focusing on physical contact for conjugation, demonstrating the necessity of direct bacterial interaction.