Lecture 16_ Bacteriophages & Transposons

Introduction to Genes and Phage Technology

• Gene Introduction: Phages can carry genes that provide functions beneficial to the host cell.

• Gene Disruption: Refers to the insertion of phage DNA into the middle of a host gene, disrupting its function.

• Transduction: Movement of genetic DNA (gDNA) from one strain of bacteria to another (not specifically phage DNA).

  • Specialized Transduction: Conducted by lysogenic or temperate phages.

  • Generalized Transduction: Conducted by lytic phages.

Types of Bacteriophages

• T-even Phages: A group of well-characterized bacteriophages.

• Undergo a lytic cycle, meaning they infect, replicate, and burst the bacterial cell to spread.

• Used in molecular biology research, particularly in understanding DNA replication and gene expression.

• Lambda (λ) Phage: A temperate bacteriophage that can switch between lytic and lysogenic cycles.

  • Prophage: Viral phage integrated into the bacterial genome.

    -It remains dormant but can be reactivated under stress (e.g., UV light).

  • Lysogen: A bacterium that carries phage DNA within its genome.

    -The phage DNA replicates with the bacterial genome without killing the host.

    -Example: E. coli can carry λ phage DNA as a lysogen.

Learning Objectives

• Understanding Phage and Transposons: Focus on topics such as transduction, transposition, and recombination.

Bacteriophages Characteristics

• Various types include:

  • Single-stranded (ss-) and double-stranded (ds-) RNA.

    -Single-stranded RNA (ssRNA)

    • Can be positive-sense (+) (acts like mRNA) or negative-sense (-) (needs to be transcribed first).

    • Example: Coronaviruses (SARS-CoV-2).

  • Double-stranded RNA (dsRNA)

    • Found in some bacteriophages and animal viruses.

    • Example: Rotaviruses.

  • Single-stranded (ss-) and double-stranded (ds-) DNA.

    -Single-stranded DNA (ssDNA)

    • Less common; requires conversion to dsDNA before replication.

    • Example: Parvoviruses.

  • Double-stranded DNA (dsDNA)

    • Most bacteriophages have dsDNA genomes (e.g., T4 phage, Lambda phage).

    • Common in viruses affecting bacteria and eukaryotes.

• Effects on Bacterial Genes: For example, Staphylococcus aureus can have its production of beta hemolysin disrupted by phages.

• Some bacteriophages integrate into the bacterial genome (prophage stage).

• In S. aureus, certain phages insert into the beta-hemolysin gene (hlb), disrupting its function.

• This prevents the bacterium from producing beta-hemolysin, a toxin that breaks down red blood cells.

• This interaction can affect virulence, immune evasion, and bacterial survival.

Phage Replication

• Always Lytic: These phages only undergo the lytic cycle, meaning they always kill the host bacterium.

  • Features approximately 100 genes.

  • Complex virion structure with head, tail, and tail fibers.

  • Example: T4 bacteriophage, which infects E. coli.

  • "Temperate phage": capable of switching between lytic and lysogenic cycles.

  • In the lysogenic cycle, the phage genome integrates into the host chromosome as a prophage.

  • The bacterium carrying the prophage is called a lysogen.

  • If triggered (e.g., UV radiation, stress), the phage switches to the lytic cycle, destroying the host.

  • Example: Lambda (λ) phage in E. coli.

Phage Burst and Development Stages

• Phage Burst: Sudden appearance of phage particles after cell lysis.

  -Marks the end of the lytic cycle when new virions escape to infect other bacteria.

• Example": T4 bacteriophange lyses E.coli releasing 100-200 new phanges

• Eclipse Phase: No phage particles are visible in the host cell due to the phases of assembly. When phage components are being made but not yet assembled

• Example: Phange DNA and proteins are synthesized but no complete virions exit yet.

• Intracellular Accumulation Phase: Phage particles are synthesized inside the host but not yet released.

T-even Lytic Cycle Steps

1. Infection: Phage attaches to a specific receptor on the bacterial cell surface.

   -Example: T4 phage binds to E. coli using tail fibers.

2. DNA Injection: Phage injects its genetic material (DNA or RNA) into the bacterial cell.

   -The empty capsid remains outside while the phage genome enters

3. Early Infection: The phage takes control of the host cell. Phage DNA starts replicating, using bacterial resources.

4. Late Infection: Structural components (capsid, tail, tail fibers) are synthesized. Host ribosomes produce phage proteins.

5. Phage Assembly: New phage particles are built inside the host. DNA is packaged into capsid heads, and tails attach.

6. Lysis: The host cell bursts open, releasing new phages (100-200 per infected cell).

   -These phages infect new bacterial cells, restarting the cycle.

Types of Regulatory Proteins in Lysogeny

• cI and cro Proteins: Control the decision to maintain either the lysogenic or lytic cycle.

  • cro: Acts as a repressor of cI and an activator of lytic genes.

• Phagemids and Cosmids:

  • Phagemids: Can integrate into and exit the genome, carrying additional DNA.

  • Cosmids: Maintain as an extra-chromosomal circle, can replace segments of the genome with target DNA.

• Why it matters?

  • Helps in understanding bacterial infections & virus replication

  • Used in phage therapy to fight antibiotic-resistant bacteria.

  • Essential in biotechnology & genetic engineering.

Lysogeny Mechanisms

Bacteriophages, especially temperate phages like lambda phage can switch between the lysogenic and lytic cycles. This switch is controlled by key regulatory proteins.

• Genetic Switch Control: When a phage integrates into the bacterial genome, it introduces stability by preventing immediate cell destruction.

  • This ensures the phage DNA is preserved within the host until conditions favor the lytic cycle

• Induction of Lytic Cycle: The transition from lysogeny to lysis can be triggered by:

  - Physical stress (UV light, heat shock, etc.)

  -Chemical agents (DNA-damaging compounds)

• Controlled by two key regulatory proteins:

  -cl protein (Lysogenic control)

  -Cro protein (lytic control)

• cI Functions: Blocks lytic genes, keeping the phage dormant in the bacterial genome.

  -Self-reinforcing-it enhances its own expression to maintain lysogeny.

• cro Functions: Inhibits cl expression, promoting the switch the lytic cycle.

  -Allows phage replication and bacterial cell lysis.

• Why this matters?

  • Explains how phages decide between dormancy and destruction

  • helps in phage therapy and bacterial gene regulation studies.

  • Shows how stress signals can reactivate dormant viruses (like herpes and HIV in humans).

Bacteriophage Lambda Overview

• Model System for Lysogeny: Lambda phage is widely studied for understanding bacterial behavior and genetics.

  -It helps researchers explore gene regulation, genetic switches, and bacterial behavior in lysogeny.

• Example: The cI vs. cro regulatory system controls whether λ phage remains dormant or enters the lytic cycle.

• Cloning System: Lambda phage is used as cloning vector because it can carry large DNA fragments (~15-20 kb)

  -Scientists modify λ phage to insert foreign DNA into bacterial genomes for genetic engineering.

• Example: Used in genomic libraries to store and manipulate large DNA sequences.

• Specialized Transduction: Unlike generalized transduction, which transfers random DNA, specialized transduction involves targeted integration.

• λ phage integrates at a specific site in the bacterial genome and when excised, may carry adjacent bacterial genes.

• This allows the exchange of specific genetic material between bacteria

• Example: transfer of galactose metabolism genes in E.coli

Transduction Mechanisms

• Transduction is a process where bacteriophages (viruses that infect bacteria) transfer chromosomal DNA between bacterial strains. it plays a key role in bacterial evolution, gene exchange, and biotechnology.

• Specialized Transduction:

  • Targeted DNA transfer using specific phage integration sites in the bacterial genome.

  • Occurs when a temperate phage (Lambda phage) integrates into a bacterial chromosome and later excises incorrectly, carrying adjacent bacterial genes.

  • May involve defective phages, which lack necessary genes to produce new viruses but still transfer bacterial DNA.

  • Example: λ phage in E. coli can transfer galactose metabolism genes (gal) during excision.

• Generalized Transduction:

  • Random DNA transfer without prior integration into the bacterial genome.

  • Occurs when a lytic phage accidentally packages bacterial DNA instead of its own genome during replication.

  • Can transfer any bacterial gene, making it valuable for mapping bacterial genomes and isolating mutations.

  • Example: P22 phage in Salmonella and P1 phage in E. coli facilitate genetic recombination.

Transposons Overview

Transposons or “jumping genes,” are mobile genetic elements that move within genomes, playing a key role in genetic variation, evolution and antibiotic resistance spread.

• Transposons: Naked DNA genetic elements capable of moving within genomes. Found in bacteria, archaea, and eukaryotes. Can disrupt genes or regulate gene expression upon insertion.

  • Insertion Elements: Short DNA sequences flanking transposons. Act as recognition sites for transposition. Contain only genes needed for movement unlike complex transposons that may carry resistance genes. Located at the ends of transposons.

  • Conjugative Transposons: move between bacterial cells through conjugation. Contribute to genetic diversity and can spread antibiotic resistance.

  • Example: Tn916 transposon in Enterococcus faecalis carries tetracycline resistance genes.

• Transposase: Cuts and moves transposons within or between genomes.

  • Recognizes insertion sequences and facilities DNA rearrangement.

  • Essential for transposon activity in gene regulation and genome evolution.

• Tn-seq (Transposon Sequencing): A high throughput method to track genetic changes caused by transposons.

  • Helps identify essential genes and study bacterial adaptation

  • Example: Used to study virulence factors in pathogens.

Transposition Mechanisms

Transposons or jumping genes, can relocate within a genome, altering gene function and contributing to genetic variation.

Jumping Ability: Transposons can hop between different locations within a genome.

-Movement can activate or inactivate genes, affecting bacterial evolution and antibiotic resistance.

• Site-specific Transposition: Target selection varies in transposition, with some transposons inserting into specific sequences, while others target a broad range of sites.

• Types:

  • Replicative: A copy of the transposon is inserted at a new location while the original stays in place.

  • Results in multiple copies of the transposon within the genome.

  • Example: Tn3 transposon in bacteria.

  • Non-replicative: The transposon physically moves to a new site without leaving a copy behind.

  • Causes disruptions in DNA sequences, impacting gene regulation.

  • Example: 1S10 transposon in E coli.

R Plasmids and Antibiotic Resistance

• R Plasmids: Carry elements that provide antibiotic resistance to bacteria

  • Often encoee enzymes that break down antibiotics (ex. lactamse for penicillin resistance)

  • Can spread between bacteria via conjugation, leading to multi-drug resistance

  • Resistance elements are often found within transposons. Allowing them to jump between plasmids or chromosomes.

    • Example: Tn5 transposon carries kanamycin resistance in bacteria.

• Transposon Behavior in Experimentation:

  • Experimenting with Staphylococcus aureus isolates to determine the source of antibiotic resistance

  • Testing Methods:

    • UV radiation exposure: Induces lytic phase in bacteriophages, helping determine if resistance genes are carried by a phage or plasmid.

    • Plasmid curing experiments: Treating bacteria with compounds (e.g., acridine orange) that remove plasmids to test if resistance disappears.

    • PCR and sequencing: Identifies specific resistance genes and their location in the genome.