Bacteriophage and CRISPR Notes

Bacteriophage

• Viruses that infect bacteria.

• Most have dsDNA but can have RNA as a genome.

• Some rely on the host's DNA/RNA polymerases.

Bacterial Viral Infections

• Virulent phage: one reproductive choice.

  • Multiplies immediately upon entry.

  • Lyses bacterial host cell.

• Temperate phages: have two reproductive options.

  • Reproduce lytically as virulent phages do.

  • Remain within host cell without destroying it.

    • Many temperate phages integrate their genome into the host genome becoming a 'prophage' in a 'lysogenic bacterium' in a relationship called lysogeny.

Lytic and Lysogenic Cycles

• Lytic cycle: Phage injects its DNA into the cytoplasm, directs the synthesis of many new phages, cell lyses and releases the new phages.

• Lysogenic cycle: Phage DNA integrates into the host chromosome (becoming a prophage), prophage DNA is copied when the cell divides, exposure to stress such as UV light triggers excision from the host chromosome.

Lysogenic Conversion

• Temperate phage changes phenotype of its host.

  • Bacteria become immune to superinfection.

  • Change host surface.

  • Phage may express pathogenic toxin or enzyme, making the host a pathogen (e.g., Cholera, Diphtheria).

• Two advantages to lysogeny for the virus:

  • Phage remains viable but may not replicate.

  • Multiplicity of infection ensures survival of the host cell.

• Under appropriate conditions, infected bacteria will lyse and release phage particles.

  • Occurs when conditions in the cell cause the prophage to initiate synthesis of new phage particles, a process called induction.

Bacteriophage T4: A Virulent Bacteriophage

• Phage life cycle culminates with host cell bursting, releasing virions.

• Steps:

  • Adsorption to receptor on E. coli outer membrane.

  • Tail sheath lysozyme/central tube pierce the cell wall.

  • Viral nucleic acid is injected into host cell through tube.

Adsorption

• Attachment of phage onto host.

• Tail fibers recognize receptor protein on the cell surface.

• Baseplate settles down on the surface.

• Shape change of baseplate and tail – Tail goes from 24 to 12 rings.

DNA Entry

• Central tube is pushed through the cell wall.

• Baseplate contains lysozyme.

• Linear DNA is extruded from the head and into host.

Bacteriophage T4 Life Cycle

• 0 min: DNA ejection

• 2 min: Early mRNA made

• 3 min: Phage DNA replicated

• 5 min: Host DNA degraded

• 9 min: Head and tails made

• 12 min: Heads filled

• 13 min: Virions formed

• 15 min: Host cell lysis

Bacteriophage T4 Life Cycle Details

• Transcription -> early mRNA.

  • Results in production of viral-encoded DNA-dependent DNA polymerase.

• Viral DNA bidirectional replication begins at several origins.

• Transcription -> late mRNA.

  • Translation of capsid and lysis proteins.

• Temporal transcription regulated by:

  • Alternative E. coli polymerase factors induced by virus.

  • Early viral gene products stimulate transcription of some late viral genes.

  • Genes with related functions are usually separated and clustered together.

    • Early gene transcribed counterclockwise.

    • Late genes transcribed clockwise.

• Inhibits the transcription of host genes.

The T4 Genome/DNA

• A large proportion of the genome codes for replication-related products including:

  • Protein subunits of its replisome.

  • Enzymes needed for DNA synthesis.

    • Synthesis of hydroxymethylcytosine (HMC), a modified nucleotide replacing cytosine in T4 DNA.

    • HMC is then chemically modified by glucosylation, protecting T4 phage DNA from E. coli restriction enzymes.

      • Enzymes that cleave DNA at specific sequences.

      • Restriction is a bacterial defense mechanism used against bacteriophage infection.

T4 DNA Is Terminally Redundant

• Base sequence repeated at both ends.

• Allows for the formation of concatamers.

  • Long strands of DNA consisting of several units linked together.

  • Structure allows for cleaving of the genome for viral progeny packaging.

  • Genome is slightly longer than the T4 gene set; each genome unit begins with a different gene.

Terminally Redundant Circular T4 Genome

• Replication yields progeny molecules with single-stranded 3' ends.

• Homologous recombination between 6 to 10 progeny molecules creates a concatemer.

• The concatemer is cleaved as DNA is packaged in phage heads, resulting in circularized genomes.

Assembly/Release of T4 Phage Particles

• Complex self-assembly process.

  • Involves viral proteins and host cell factors for capsid assembly.

• Set of proteins that package DNA.

  • Packasome moves DNA into phage head.

  • Terminase complex generates double-stranded ends, cuts concatemer, and pushes DNA into head.

• Assembly followed by release.

  • In T4 - E. coli system, $150$ viral particles are released.

  • Two proteins are involved in the process:

    • T4 lysozyme attacks the E. coli cell wall.

    • Holin creates holes in the E. coli plasma membrane.

Bacteriophage Lambda: A Temperate Bacteriophage

• Phage lambda (λ) can enter either the lytic or lysogenic cycle upon infection of E. coli.

  • Lysogenic: dsDNA becomes prophage, integrated into the host’s chromosome.

  • Upon induction, the viral genome is excised and the lytic cycle begins.

Lambda Phage DNA

• Linear ds DNA genome with cohesive ends.

  • Circularizes upon injection into host cytoplasm.

  • $40$ genes, genes clustered together by function.

  • Transcription from different promoters determines if the lytic cycle or lysogeny occurs.

Regulatory Proteins Determine Lysogeny or the Lytic Cycle

• Function as repressors, activators, or both.

  • Regulate transcription, termination, and antisense RNA molecules.

• cII activator plays a pivotal role in determining if λ will establish lysogeny or the lytic cycle.

  • cII levels high early in infection – lysogeny.

  • cII levels not high early in infection – lytic cycle.

Lytic or Lysogenic Pathway: High cII Levels

• CII is made early.

• If CII levels get high enough:

  • int gets made (needed for integration).

  • CII also increases CI levels, which represses all other genes.

λ Phage and High cII Levels

• Increases int gene transcription.

  • Integrase catalyzes integration of λ into the host genome; lysogeny is established.

• Increases transcription of cI gene (λ repressor).

  • λ repressor represses all transcription except its own.

  • Binds to PRM promoter and activates transcription of cI; therefore, lysogeny is maintained.

Integrase function

• λ integrates at the att site on the host chromosome.

  • The phage genome has a corresponding site.

• Integrase catalyzes site-specific recombination.

Lytic or Lysogenic Pathway: Low cII Levels

• CII gets degraded by host.

• CIII protects CII.

• Cro is repressed by CII.

• Cro inhibits CIII and CI.

• Cro makes Q, which is necessary for the lytic cycle.

λ Phage and Low cII Levels

• cII is quickly degraded by a host enzyme, HflB, unless it is protected by viral cIII.

  • cIII is made at the same time as cII.

• If cII is not protected, protein Cro increases.

  • Cro is a repressor that inhibits transcription of cIII and cI genes, further decreasing cII and λ repressor.

  • Cro is also an activator that increases transcription of itself (Cro) and the regulatory protein Q.

• Q activates genes needed for the lytic cycle.

How Does Induction Reverse Lysogeny?

• Triggered by a drop in λ repressor levels, due to UV light or mutagenic chemicals.

• DNA damage alters host cell RecA protein, interacting with λ repressor, causing the repressor to cleave itself.

• cI transcription decreases, λ repressor levels are reduced further.

• Transcription increases:

  • xis gene, excisionase increases and binds integrase, reversing integration; λ phage freed from host chromosome.

  • Cro protein levels increase, blocking synthesis of λ repressor.

  • Protein Q increases, and the lytic cycle proceeds.

Other Temperate Phages

• Most exist as site-specific integrated prophages.

• Bacteriophage Mu: transposition allows random integration sites; a repressor protein inhibits lytic growth.

• E. coli phage P1: the lysogenic cycle occurs in the absence of integration; P1 and E. coli replicate together.

Bacterial Immune System

• CRISPR

Immunity

• Innate (non-specific)

  • Restriction enzyme

  • Receptor modifications

• Adaptive (learning)

  • Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

CRISPR

• Protects against viruses and other mobile genetic elements.

• Found in 85% of archaea and 45% of bacteria.

• Array of short repeats separated by unique spacers, often derived from viruses.

• Requires CRISPR associated (cas) genes.

• RNA binds to foreign DNA to target its degradation.

CRISPR Details

• Repeats are $21-48$ bp.

• Spacers are $26-72$ bp.

• The number of spacers can vary widely, with new spacers added every time a cell survives a viral attack.

• Genomes can have 1 or more CRISPR loci.

CRISPR Defense

• Adaptation: Insertion of new spacers into CRISPR locus.

• Expression: Transcription of CRISPR locus and processing.

• Interference: Detection and degradation of foreign DNA.

Adaptation in Detail

• Provides the genetic memory - insertion of new spacer.

• Cas1 and Cas2 are nucleases that form a dimer.

• Protospacer adjacent motif (PAM): Motif in target sequence crucial for self/non-self recognition.

Adaptation: Protospacer Selection

• Protospacer Selection:

  • Recognized by Cas proteins.

  • DNA may be copied or cut directly out of the source.

• Generation and insertion of spacer material:

  • Cas1 nicks CRISPR array in E. coli.

  • DNA is inserted in chromosome.

Expression in Detail

• Transcription of CRISPR-Cas to generate an RNA-protein guide complex.

• Transcribe CRISPR locus – Makes long RNA.

• Process the RNA with Cas ribonucleases.

  • Forms CRISPR ribonucleoprotein complex.

  • Cut into single spacer-sized RNA.

Interference in Detail

• crRNA-Cas locates protospacer to trigger target degradation, performed by Cas-specific nucleases.

• Some require PAM and perfect protospacer-crRNA complementarity, stopping systems from attacking itself (Types I, II, III).

Anti-CRISPR Mechanisms

• Viral mutations: DNA is no longer a match to the spacer, interfering with PAM recognition.

• Anti-CRISPR proteins: Viral proteins that specifically inhibit CRISPR-Cas complexes.

Applications of CRISPR

• Diagnosis and epidemiology: PCR-based approach to genotyping.

• Dairy industry: Make strains that have CRISPR toward problematic phages.

• Eukaryotic genetic research: Type II system.

Type II CRISPR

• Cas9 assists in adaptation, crRNA processing, and cleaves target DNA.

  • Cleavage requires crRNA and tracrRNA.

Genetic Applications

• crRNA and tracrRNA can be combined to a single guide RNA (sgRNA).

• Cas9 produces a single double-stranded break in the target DNA.

  • Can be recombined into the host genome using 2 host repair mechanisms.

CRISPR: Ethical Considerations

• "With great power comes great responsibility".

• Novel CRISPR-derived ‘base editors’ surgically alter DNA or RNA, offering new ways to fix mutations.

  • Science comment about Science and Nature papers.

• Correction of a pathogenic gene mutation in human embryos – Nature. (Reference to a Nature publication).