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 hot 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 cells 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 ca 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 a 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, log 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 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 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 releases 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 with 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