Lecture #18 | Bacteriophage

Prokaryotic Viruses

Must bind to host cell receptor, must cross a cell wall (2 in gram negative hosts) but not damage cell initially, must utilize the host's cellular machinery (nucleotides, amino acids, ATP) to replicate, exit through cell wall by usually lysing host cells.

Bacteriophage

Viruses that attack bacteria but do not harm eukaryotes. Called a phage (means to eat).

Life cycle of a Bacteriophage

1. Attachment to host cell proteins like bacterial cell receptors which are normally used for bacterial purposes like sugar uptake, conjugation 2. Injects genome through cell wall to cytoplasm in a very strong injection. The phage capsid remains outside and is attached to the cell surface as a ghost.

Bacteriophage replication

Requires a host cell (like all viruses) for reproduction.

Bacteriophage needs for replication

Host recognition and attachment, genome entry, assembly of virons, exit and transmission.

How does a bacteriophage attach to a host cell?

Contact and attachment are mediated by cell-surface receptors.

Cell-surface receptors

Proteins that are specific to the host species and bind to a specific viral component. They are normally used in bacterial cells for important cellular functions like sugar uptake.

What does this image depict?

A bacteriophage is injecting its genome through a cell’s wall. While the genome is the part being injected inside the cell, the phage capsid remains outside and is attached to the cell surface.

What are the two types of life cycles that a bacteriophage can undergo?

Lytic cycle and lysogenic cycle.

Lytic cycle

Bacteriophage quickly replicates, killing host cell.

Lysogenic cycle

Bacteriophage is quiescent (inactive) and integrates into cell chromosome as a prophage (integrated into circular chromosome). It can reactivate and become lytic.

What determines the decision between lytic and lysogenic cycles?

Environmental cues as events that threaten a host cell survival trigger a lytic burst.

Bacteriophage life cycle in the lytic phase

1. A bacteriophage uses a cell's components to synthesize capsids 2. Progeny phages are assembled 3. The bacteriophage exit from the cell through either lysis or slow release.

How do bacteriophages exit the cell in the lytic phase?

Either lysis (a protein is made that depolymerizes peptidoglycan and bursts host cell to release new phage) or slow release (filamentous phages can extrude individual progeny through the cell envelope).

How are bacteriophages cultured?

1. A top agar is made by mixing a diluted phage with agar and E. coli 2. Serial dilutions are made 3. The top agar is added onto plates with a bottom agar 4. Plates are incubated overnight as the E. coli grows and the phage infects the E. coli cells and multiply forming plaques which contain about 10^6 phage from one parent.

How to interpret plates after bacteriophage culture?

Cloudy areas are growth of E. coli while clear areas are plaques, showing where the E. coli was killed by the phage.

What are plaques when culturing bacteriophages?

Areas that the E. coli is killed by the phage and are formed upon the lysis of E. coli cells. One plaque = infection of one virion (quantified in plaque forming units (pfu)).

Coliphages

Viruses that infect E. coli, divided into T4 and lambda (both have double-stranded linear DNA genome).

T4 Phage

A capsid head, contractile tail, lytic cycle (intemperate).

λ phage

Capsid head, non-contractile tail, lysogenic (temperate).

Structure of a bacteriophage T4

Complicated structure with 170 genes, 10 different capsid protein types, tail fibers bind to host cell, long tail injects DNA.

What type of receptor does a T4 bacteriophage bind to?

A OmpC porin which is an outer membrane protein.

Life cycle of a T4 phage

Takes approximately 25 minutes 1. Absorption and penetration 2. Early mRNA synthesis 3. DNA replication 4. Late mRNA synthesis and formation of new viral particles 5. Lysis.

Absorption and penetration of a T4 phage

The phage particle attaches itself to the outer membrane of the E. coli and injects its genome. It essentially drills through the cell wall(s) until it reaches the cytoplasm, then it injects its genome.

Early mRNA synthesis in T4 bacteriophage

Early genes are transcribed and translated to proteins including nucleases to cleave host DNA and proteins for phage DNA replication, these take control of the cell and destroy the cell chromosome.

DNA replication in T4 bacteriophage

Genome is injected as linear but forms a circle with terminal repeats where the cells nucleotides are used to replicate genome using a rolling circle replication (continuous replication of many copies of genome). This concatemer, which has several genomes linked together, is the cut according to DNA lengths with an offset so that individual linear genomes have slight overlaps.

Formation of virus heads in T4

DNA that is slightly longer than phage genome (103%) is packaged into virus heads, as the sequence begins and ends at different points in different heads.

Late mRNA transcription in T4

Capsid particles are made before bacteriophage genome is encapsulated into a virus head. These include tail fibers, head, long tail, which are then assembled into phages. Lysis proteins are also made which destroy the cell wall and release the progeny.

Lysogenic bacteriophage

There is a stable relationship between the host cell and a temperate phage where the phage is neither multiplying nor lost. Done by integrating into host DNA, which is replicated as the host cell grows. Can enter lytic cycle when it fears the host cell may die.

Lysogen

A bacterium that is harboring a prophage in its viral genome. Immune to superinfection, where there can only be a single lysogenic virus in a host cell.

DNA of a λ phage

Double stranded and linear DNA when in phage head with cohesive ends (cos sites: determine where the genome is cut and packaged) which are complementary single stranded stretches. When in host cell, the DNA circularizes once injected, as tandem repeating ends combine in the host to give a circular configuration of the ligated cos site.

λ phage receptor

Binds to a maltose porin in outer membrane of E. coli.

Induction of Lytic Cycle from Lysogenic Cycle

Stimulated by DNA damage of the host cell from UV light, chemical mutagens. This activates a SOS response which initiates transcription of xis for excision of the prophage from the bacterial genome.

How is λ genome replicated?

Begins with θ replication (circle-to-circle) at the beginning, which is initiated at the ori site and has bidirectional replication. Then it is replicated in a rolling circle replication to create long chains of concatenated genomes. Genome is cut at the cos site.