Topic B - Bacteriophage Life Cycle

Phases of Lytic Life Cycle

1. Adsorption - Bacteriophage binds to receptor on bacterial cell surface

2. Injection - Injection of genetic material into the cell

3. Early gene expression

4. DNA replication

5. Late gene expression

6. Morphogenesis

7. Host Cell Lysis

Adsorption

Phage binds to specific receptors on the bacterial surface

-Receptor binding protein found on tail (for tailed phages)

-Every phage targets its own receptor

-Receptor not the intended function, just exploited by the phage

Examples of receptors on Gram-negative bacteria:

1. Outer-member proteins

2. LPS

Injection

Phage genome is passed from the capsid → through the bacterial envelope → into the cytoplasm of the host cell

-Mechanisms of Injection:

1. Contractile tail

2. Enzymes digest peptidoglycan

3. Outer membrane transport proteins

4. Pilus retraction

Injection T4 Genome into E.coli

1. Tail fibers bind to LPS

2. Tail fibers retract

3. Tail pins bind tightly to bacterial cell surface → stable association with bacterial cell surface

4. Tail sheath contracts → tail tube shoots out through membrane into peptidoglycan, end of the tube contains lysozome

5. T4 lysozyme digests small hole in PG → tail tube moves past PG through the inner membrane

6. DNA is injected into cytoplasm

Early Gene Expression

Promoters of early genes look like host cell promoters

-Transcribed by host RNA polymerase

Early genes encode;

1. Proteins that prevent host-cell gene expression

2. Enzymes that degrade bacterial chromosome

3. Enzymes for phage DNA replication (some) (ex; enzymes that make hydroxylmethylcytosine)

4. Transcription factors needed for transcription of late gene promoters

Phage DNA Replication

Uses combination of host cell and phage-encoded DNA replication proteins

-hundreds of copies of phage DNA produced

Late Gene Expression

Promoter of late genes do not look like host cell promoter

-Require;

1. Phage transcription factors to help host RNA polymerase, or

2. Phage RNA polymerase that recognizes late promoters

Late genes encode:

-Structural proteins (capsid and tail)

-Morphogenesis proteins

-Enzymes for host cell lysis

Morphogenesis

Assembly of new phage particles:

-Capsid is assembled

-DNA is inserted

-Tail is attached

Process is different for every phage:

-Some assemble spontaneously

-Some require viral proteins to assist in packaging

Assembly of T4 Phage

1. Capsid assembled on a protein scaffold

2. Motor protein attached to capsid

3. DNA is injected into the capsid (ATP dependent)

4. Scaffold and motor proteins are removed

5. Tail assembly and attachment occurs spontaneously

Host Cell Lysis

Bacterial host cell bursts open to release new phage particles:

-Burst size - average number of phage released per bacterial cell

Requires phage enzymes encoded by late genes:

1. Holin - Disrupts cytoplasmic membrane and allows release of enzymes that degrade peptidoglycan

2. Lysozyme/Endolysin - breaks bond between sugars in peptidoglycan

3. Spanin (gram-negative hosts) - creates holes in the envelope by fusing the inner and outer membranes

• One end of Spanin anchors into the outer membrane, the other anchors into the inner membrane

• Spanin pinches together, fusing the two membranes together; fusion creates a hole

Regulation of the Lytic Cycle

Phage gene expression is controlled by a regulatory cascade

-Early genes active expression of late genes

-Late genes repress expression of early genes

Regulation occurs at the promoters

Prophage, Lysogen and Lysogeny

Prophage - Phage DNA integrated into the host chromosome

Lysogen - Bacterium containing one or more prophage

Lysogeny - Lysogenic life cycle

Stages of the Lysogenic Life Cycle

1. Adsorption

2. Injection

3. DNA circularizes

4. Lysogeny decision

5. Integration

6. Replication

7. Induction of the lytic cycle

DNA Circularizes

Linear phage genome contains complementary single-stranded ends called Cos Sites

-These will base-pair to each other to circularize the linear phage DNA molecule

Lysogeny Decision

Phage use chemical signals to assess the conditions within the host cell

-If conditions are bad, phage start the lytic cycle

• DNA damage

• Few phage in the environment (low MOI)

-If condition are good, phage starts lysogeny

• Host cell is healthy

• Lots of phage in the environment (high MOI)

MOI - Multiplicity of Infection, ratio of bacteria to bacteriophage

Lysogency Decision - Lambda Repressed (cl)

Lambda Repressor (cl) is expressed if conditions are favourable for lysogeny

-Repress the expression of lytic genes from the phage genome by binding to promoters of early genes and block transcription

-Prevents the lytic cycle from starting

Integration

-Integration occurs through site-specific recombination

Integrase - Enzyme that facilitates integration of the phage DNA into the host Bacterial chromosome

-Site-specific DNA recombinase

Catalyzes recombination between the attP in the phage genome and attB in the bacterial chromosome

-NOT homologous recombination because the two DNA sequences are NOT the same

Integration of Lambda Phage into E.Coli Chromosome

attB is located in a non-essential region of bacterial chromosome so integration doesn’t result in an insertion mutation

-After integration, attB and attP sites are halved into two new combinations

-Hybrid sequences (attpB/P and attp/B) found on either side of the prophage

Attp vs Cos Site

Attp site is for inserting the circularized phage genome into the host chromosome

Cos site is for circularizing phage DNA during infection

Replication

Prophage DNA is replicated along with the bacterial chromosome as the lysogen divides

-uses bacterial host’s energy and replication machinery

-Every daughter cell will contain the prophage

Over many generations, lysogen can take over the bacterial population

Induction of the Lytic Cycle

Phage DNA is removed from the bacterial chromosome and lytic cycle starts

-Triggered by cell stress, repression of the lytic genes by cl is lost

-Phage genes are expressed, including:

1. Excisionase - Enzyme that helps cut prophage DNA out of bacterial chromosome

2. Early genes of the lytic cycle

DNA Damage and Prophage Excision

DNA damage can induce prophage excision

-DNA damage results in ssDNA fragments

-RecA protein binds to ssDNA creating a protease

-RecA-ssDNA cleaves the cl repressor

-Integrase (int) and excisionase (xis) are expressed once cl is removed

Integrase in the Lytic Cycle

Integrase can’t recognize attB/P or attP/B

-Integrase and excisionase form a complex

1. Excisionase helps integrase bind to attB/P and attP/B

2. Promotes site-specific recombination between attB/P and attP/B

Phage DNA is excise and recircularized

Final Stage of Induction

Once prophage is removed from the bacterial chromosome, normal lytic phage replication begins

-Early gene expression → DNA replication → Late gene expression → Morphogenesis → Host cell lysis

Lysogenic Conversion

Some prophage contain additional genes not involved in lysis or lysogeny

-These genes can alter host characteristics/capabilities

Examples: Cholera toxin and Shiga toxin

Cholera Toxin - Lysogenic Conversion

-Nonpathogenic vibrio is infected by two phages to produce cholera toxin

TCP Phage - Encodes genes for the toxin co-regulated pilus (TCP pilus)

CTXo Phage - Uses the TCP as a receptor, will encode genes for cholera toxin production

-Turns the nonpathogenic vibrio into a pathogenic strain

Shiga Toxin - Lysogenic Conversion

Encoded on the Stx phage genome

-Expression of shiga toxin is repressed by a cl-like protein during lysogeny

Superinfection Immunity

Existing prophage prevents another phage from infection a lysogen (superinfection)

-Prevents entrance or replication of a superinfection phage

-Can modify cell-surface receptors to prevent phage binding or entry

Repressors expressed by prophage can prevent gene expression from superinfecting phage

Types of Phages

Virulent Phage/Lytic Phage - Phage that undergoes only lytic growth

-Ex; T4 phage

Temperate Phage - Capable of both lytic growth and lysogeny

-Ex; bacteriophage lambda

Efficiency of Plating (EOP)

Fraction of phage particles in a stock that can form a plaque

EOP = 1 → All phage form plaques

Lytic Phage - EOP is high, all correctly formed phages should form a plaque

Lysogenic Phage - EOP is much less than 1, most phage integrate, few plaques form spontaneously

Multiplicity of Infection (MOI)

Ratio of phage:bacteria in a culture

-One bacterium can be infected by more than one phage particle

-Determines how many bacterial will be infected in a culture:

MOI < 1 = Not all bacteria will be infected

MOI > 1 = each bacterium will be infected by multiple phage

Co-Infection

Infection of a susceptible bacterial culture with two different mutant phage at a high MOI

-some bacteria will be infected by both phages

-two different mutant phage will interact in host cell → Allows the study of mutations

Recombination - interaction of mutant genes

Complementation - interaction of mutant proteins

Host Range

Type of host bacteria that can be infected

-Phage infection shows host specificity due to

1. Bacterial and phage receptor compatibility

2. Receptor modification systems of bacteria

3. Cell machinery compatibility

Ex; Phage P22 infects Salmonella but not E.coli

Host Range Mutants

Extended Host Range Mutant - Mutant phage that can infect bacteria that wildtype phages cannot

Phage Therapy

Phage infect bacteria causing illness, which are then lysed/killed

Advantages of Phage Therapy

-Works against antibiotic resistant bacteria

-Specific — will not destroy microbiome

-Replicate during treatment

-Very few side effects

Disadvantages of Phage Therapy

-Specific - need to know exactly what is causing the infection

-Bacteria can evolve phage resistance

-Hard to claim intellectual property

-PR problems