Phage Strategies

Presented by

Amith ReddyEastern New Mexico University

What is a bacteriophage ?

A bacteriophage is a virus

that infects and replicates

within bacteria.

Eg: Lambda phage, T4, T7

27.1 Introduction

27.1 Introduction

• bacteriophage (or phage) – A bacterial virus.

• lytic infection – Infection of a bacterium by a phage that

ends in the destruction of the bacterium with release of

progeny phage.

• lysis – The death of bacteria at the end of a phage infective

cycle when they burst open to release the progeny of an

infecting phage (because phage enzymes disrupt the

bacterium’s cytoplasmic membrane or cell wall).

27.1 Introduction

• virulent phage – A bacteriophage that can only follow the

lytic cycle.

• prophage – A phage genome covalently integrated as a

linear part of the bacterial chromosome.

• lysogeny – The ability of a phage to survive in a bacterium

as a stable prophage component of the bacterial genome.

27.1 Introduction

FIGURE 01: A phage may follow the lytic or lysogenic pathway

27.1 Introduction

• temperate phage – A bacteriophage that can follow the lytic

or lysogenic pathway.

• integration – Insertion of a viral or another DNA sequence

into a host genome as a region covalently linked on either

side to the host sequences.

• excision – Release of phage from the host chromosome as

an autonomous DNA molecule.

27.1 Introduction

• induction of phage – A phage’s entry into the lytic

(infective) cycle as a result of destruction of the lysogenic

repressor, which leads to excision of free phage DNA from

the bacterial chromosome.

• plasmid – Circular, extrachromosomal DNA. It is

autonomous and can replicate itself.

• episome – A plasmid able to integrate into bacterial DNA.

Lytic and Lysogenic cycle

http://www.youtube.com/watch?v=gU8XeqI7yts

• Ensure replication of DNA• Initiation of replication• Carries DNA polymerase• Engage in Transcription

Result: Phage mRNAs are transcribed.

Bacterial mRNA replaced by

Phage mRNA.

27.2 Lytic Development Is Divided into Two Periods

27.2 Lytic Development Is Divided into Two Periods

• A phage infective cycle is divided into the early period (before replication) and the late period (after the onset of replication).

• A phage infection generates a pool of progeny phage genomes that replicate and recombine.

FIGURE 02: Phages reproduce in lytic development

Early Phage Late Phage

* Production of Enzymes * protein synthesis

* DNA synthesis, recombination * Structural proteins

* Replication pool is created * Assembly proteins

* By the time the structural components are assembled into head and tail,

DNA replication reaches it maximum rate.

• Each group of phage genes can be expressed only when

an appropriate signal is given.

• Cascade of gene expression

– Groups of genes are turned on ( or off) at particular

times.

• Cascade of regulators

– Each set of genes contains at least one necessary for

transcription of the next set of genes.

27.3 Lytic Development Is Controlled by a Cascade

27.3 Lytic Development Is Controlled by a Cascade • cascade – A sequence of

events, each of which is stimulated by the previous one.– Transcriptional regulation is

divided into stages, and at each stage one of the genes that is expressed encodes a regulator needed to express the genes of the next stage.

FIGURE 03: Lytic development is a regulatory cascade

27.3 Lytic Development Is Controlled by a Cascade

• The early (or immediate early) genes transcribed by host

RNA polymerase following infection include, or comprise,

regulators required for expression of the middle (or delayed

early) set of phage genes.

• The middle group of genes includes regulators to transcribe

the late genes.

• This results in the ordered expression of groups of genes

during phage infection.

Fig A: Early genes switched off, when middle gene are transcribed.

Fig B: Early genes are continued to be expressed.

A B

To sum up

Stage I: Early genes transcribed by host mRNA poly.

Stage II : Production of enzymes needed for replication.

Stage III : Production of genes for building phage components.

• Active genes are regulators.

• Regulator

• 1. New sigma factor– Controls binding of DNA by recognizing specific sequence in

promoter DNA.

• 2. Antitermination factor– Reads a new group of genes.

27.4 Two Types of Regulatory Events Control the Lytic Cascade

27.4 Two Types of Regulatory Events Control the Lytic Cascade

• Regulator proteins used in phage cascades may sponsor initiation at new (phage) promoters or cause the host polymerase to read through transcription terminators.

FIGURE 06: RNA polymerase controls promoter recognition.

Fig A: Transcripts are independent. Early gene expression ends after sigma factor is produced.

Fig B: Early genes are separated form next expressed genes by terminator site.

A B

• The new genes are expressed only by extending the

RNA chain to form molecules that contain early gene

sequence at 5' end and new sequence at 3' end.

• The regulator gene that controls the switch from

immediate early to delayed early expression in phage

lambda is identified by mutations in gene N.

• Gene N can transcribe only the immediate early genes.`

27.5 Phage T7; cascade of gene expression

Class I : Immediate early type expressed by host RNA polymerase.

Class II : Expressed by phage RNA poly.

Class III : Assembly of phage particle.

T7 genome size : 38 kb.

27.5 The Phage T7 and T4 Genomes Show Functional Clustering

• Genes concerned with related functions are often clustered.

FIGURE 08: T4 genes show functional clustering

• Early genes : Transcribed by host RNA poly.

• Middle genes : Transcribed by host RNA poly

+

phage encoded products MotA & AsiA

• Middle promoter lacks consensus 35 seq and have a

binding seq for MotA.

• Phage protein (activator) compensates by making host

RNA polymerase to bind.

27.5 The Phage T7 and T4 Genomes Show Functional Clustering

• Phages T7 and T4 are examples of regulatory cascades in which phage infection is divided into three periods.

FIGURE 09: T4 genes fall into two general groups

• Both cycles start when DNA enters the host.

• Lytic cycle occurs if late genes are expressed.

• Lysogeny occurs if synthesis of lambda repressor (gene

regulator)

• This occurs by turning on cI gene. (Remember this point)

27.6 Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny

and the Lytic Cycle

• Lambda has two immediate early genes, N and cro,

which are transcribed by host RNA polymerase.

• The N gene is required to express the delayed early

genes.

• The cro gene code for prevention of expression of cI

• Three of the delayed early genes are regulators.

Phage lambda early genes• Immediate early genes:

• N– Antiterminator– acts at nut sites– Allows transcription to proceed to delayed early genes

• cro– prevents synthesis of repressor ( lytic cycle)– turns off expression of immediate early genes

Regulator genes have opposing function

• The cII-cIII : Establish the synthesis of the lambda

repressor for lysogenic pathway.

• The Q regulator : codes for antitermination factor which

allows the host RNA poly to

transcribe the late genes.

Role of Delayed Early genes

1. Helps the phage to enter the lysogeny.

2. Controls the order of the lytic cycle.

* At this point lambda is keeping open the option to

choose either pathway.

27.6 Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny

and the Lytic Cycle

• Lysogeny requires the

delayed early genes cII-cIII.

• The lytic cycle requires the

immediate early gene cro and

the delayed early gene Q.

FIGURE 10: Lambda has two lifestyles

Fig 11: Map of lambda phage, genome is 48,514 bp

* Lytic cycle genes are expressed in polycistronic transcripts from 3 promoters.

• A group of genes concerned

with regulation are surrounded

by genes needed for

recombination and replication.

• Structural genes are

clustered.

27.7 The Lytic Cycle Depends on Antitermination by pN

FIGURE 12: Lambda phage genes are organized in two transcription units.

• Initiation of transcription at PL and PR.

• N is transcribed towards left, into recombination genes.

• Cro is transcribed towards right, into replication genes.

• pN (regulator) allows transcription to continue into the delayed early genes by suppressing the terminators tL and tR.

Fig 13: Lambda has three stages of development

Lambda DNA circularizes after infection

• pN is an antitermination factor that

allows RNA polymerase to

continue transcription past the

ends of the two immediate early

genes.

• pQ is the product of a delayed

early gene and is an antiterminator

that allows RNA polymerase to

transcribe the late genes.

Result

• Transcript length is 194 bases

(6S RNA)

• When pQ is available, 6S RNA is

extended.

• Hence late genes are expressed.

• Right end has lysis genes S-R

• Left end has head and tail genes A-J.

• The late genes form a single

transcription unit starting from PR

(lies bet Q and S)

• Late transcription terminates at site

tR3.

Fig 14: Lambda phage regulatory

region

• Promoter PL and PR Operator OL and OR

• Repressor binds at operator to prevent RNA poly to initiate transcription.

• Since the seq overlaps with the promoter these seq are called PL/OL and PR/OR control regions.

* By denying RNA polymerase access to these promoters, the lambda repressor protein prevents the phage genome from entering the lytic cycle.

cI expression

• PRM-promoter region for cI– RM repressor maintenance– Weak promoter

27.8 Lysogeny Is Maintained by the Lambda Repressor Protein

• The lambda repressor, encoded by the cI gene, is required to maintain lysogeny.

• The lambda repressor acts at the OL and OR operators to block transcription of the immediate early genes.

• The immediate early genes trigger a regulatory cascade; as a result, their repression prevents the lytic cycle from proceeding.

FIGURE 15: Repressor maintains lysogeny

27.9 The Lambda Repressor and Its Operators Define the Immunity Region

• Immunity – In phages, the ability of a prophage to prevent another phage of the same type from infecting a cell.

• Virulent mutations – Phage mutants that are unable to establish lysogeny.

• Immunity regions – left & right operators + cI + Cro gene

27.9 The Lambda Repressor and Its Operators Define the Immunity Region

• Several lambdoid phages have different immunity regions.

• A lysogenic phage confers immunity to further infection by any other phage with the same immunity region.

FIGURE 16: RNA polymerase initiates at Pl and Pr but not at Prm during the lytic cycle.

Autogenous circuit

• cI – Repressor for immediate early genes– Positive regulator for cI– RNA pol cannot initiate efficiently at PRM in

the absence of cI

• Autogenous circuit– Presence of cI is necessary to support its own

synthesis

The immunity region of lambda phage

Immunity region– OL-cI- OR-cro– Specifies the repressor and the sites to which the

repressor acts

The immunity region: repressor & operators

• Virulent mutations– OL, OR, vir– vir– Grows on lysogens, vir mutations allow the

incoming phage to ignore the resident repressor and enter the lytic cycle

• Lytic Cycle– Cascade of transcriptional controls

• Lysogeny– Autogenous circuit

– Repressor binding

27.10 The DNA-Binding Form of the Lambda Repressor Is a Dimer

• A repressor monomer has two distinct

domains.

• The N-terminal domain contains the DNA-

binding site.

• The C-terminal domain dimerizes.

• Binding to the operator requires the dimeric

form so that two DNA-binding domains can

contact the operator simultaneously.

27.10 Repressor functions as a dimer

• Each domain exercise its function

separately.

• C-terminal fragment forms oligomers.

• N-terminal fragment binds the

operator.

* The dimeric structure of the lambda repressor is crucial in maintaining lysogeny.

1

DNA-binding Connector Dimerization

92 132 236

N C

111 - 113

Cleavage site

Repressor subunit

• 27 kb

• Binds to DNA as a dimer

• Uses a helix-turn-helix motif

• Cooperative binding

• Cleavage of the repressor between the two domains reduces the affinity for the operator and induces a lytic cycle.

• The lysogeny - lytic cycle switch depends on the repressor concentration

– Too high

• Impossible to induce the lytic cycle

– Too low

• Impossible to maintain lysogeny

FIGURE 18: Repressor cleavage induces lytic cycle

27.11 Lambda Repressor Uses a Helix-Turn-Helix Motif to Bind DNA

• Each DNA-binding region in the repressor contacts a half-site in the DNA.

• The DNA-binding site of the repressor includes two short α-helical regions that fit into the successive turns of the major groove of DNA (helix-turn-helix).

• A DNA-binding site is a (partially) palindromic sequence of 17 bp.

FIGURE 19: The operator is a palindrome

Half site

Repressor: helix-turn-helix motif

• Helix 3 9 aa• Helix 2 7 aa

Binding specificities

• Helix 3– Hydrogen bonds between aa of helix 3 and exposed

DNA bases.– This helix recognizes specific DNA seq (recog helix)– Specific binding

• Helix 2– Hydrogen bonds helix 2- aa and the phosphate

backbone– Nonspecific binding

Repressor binding to the operator

• The repressor binds simetrically to the site• Each N-terminal domain contacts a similar DNA

sequence

27.11 Lambda Repressor Uses a Helix-Turn-Helix Motif to Bind DNA

• The amino acid sequence of the recognition helix makes contacts with particular bases in the operator sequence that it recognizes.

Fig 22: Helix-3 determines DNA-binding specificity

• Lambda repressor and cro select different sequence in the DNA as their targets because they have different seq of aa in helix 3

• These interactions are necessary for binding, but do not control the specificity of target recognition.

repressor makes an additional contact

• Helix 1– Lysine residues make contact with G residues in the major

groove– Deletion of helix 1 reduces affinity by ~1000 fold

The operators; three repressor binding sites

The operators; three repressor binding sites

• Binding sites– 17 bases– partial symmetry– 3-7 bases A-T rich spacers

• A repressor dimer binds symmetrically at each site

How does the repressor decide where to bind?

27.12 Lambda Repressor Dimers Bind Cooperatively to the Operator

• Repressor binding to one operator increases the affinity for binding a second repressor dimer to the adjacent operator.

• The affinity is 10× greater for OL1 and OR1 than other operators, so they are bound first.

• Cooperativity allows repressor to bind the OL2/OR2 sites at lower concentrations.

Fig 25: Lambda repressors bind DNA cooperatively

cI mutants show that N-terminal region interacts with RNA polymerase

27.13 Lambda Repressor Maintains an Autoregulatory Circuit

• The DNA-binding region of repressor at OR2 contacts RNA polymerase and stabilizes its binding to PRM.

• This is the basis for the autoregulatory control of repressor maintenance.

• Repressor binding at OL blocks transcription of gene N from PL.

FIGURE 26: Repressor maintains lysogeny but is absent during the

lytic cycle

27.13 Lambda Repressor Maintains an Autoregulatory Circuit

FIGURE 27: Helix-2 interacts with DNA polymerase

• Repressor binding at OR blocks transcription of cro, but also is required for transcription of cI.

• Repressor binding to the operators therefore simultaneously blocks entry to the lytic cycle and promotes its own synthesis.

27.14 Cooperative Interactions Increase the Sensitivity of Regulation

• Repressor dimers bound at OL1 and OL2 interact with dimers bound at OR1 and OR2 to form octamers.

• These cooperative interactions increase the sensitivity of regulation.

FIGURE 29: Repressors to bind to OL3 and OR3 at higher concentrations

27.15 Sequential steps

• Lamdha DNA enters the host cell .

• RNA poly cant transcribe cI (as no repressor to aid

binding at PRM)

• First event : N and cro genes are transcribed.

• pN allows transcriptions to allow further.

• Hence cIII trancribes on left and cII on right.

* cII and cIII genes are positive regulators whose products are needed for alternative system for repressor synthesis.

27.15 The cII and cIII Genes Are Needed to Establish Lysogeny

• The delayed early gene products cII and cIII are necessary for RNA polymerase to initiate transcription at the promoter PRE.

• cII acts directly at the promoter and cIII protects cII from degradation.

• Transcription from PRE leads to synthesis of repressor and also blocks the transcription of cro.

FIGURE 30: Repressor establishment uses a special promoter

Expression from PRE

• cII activates cI transcription from PRE

• cIII prevents cII degradation

• Transcription at PRE promotes lysogeny– 5’ part of RNA anti-sense RNA for cro– cI is transcribed– cI translation from is PRE 7-8 X more efficient than

PRM

27.16 A Poor Promoter Requires cII Protein

• PRE has a typical sequences

at –10 and –35.

• RNA polymerase binds the

promoter only in the

presence of cII.

• cII binds to sequences close

to the –35 region.

Fig 31: cII enables RNA polymerase to bind to PRE

27.17 Lysogeny Requires Several Events

• cII and cIII cause repressor synthesis to be established

and also trigger inhibition of late gene transcription.

• Establishment of repressor turns off immediate and

delayed early gene expression.

• Repressor turns on the maintenance circuit for its own

synthesis.

• Lambda DNA is integrated into the bacterial genome at

the final stage in establishing lysogeny.

27.17 Lysogeny Requires Several

Events

FIGURE 33: The lysogenic pathway leads to repressor

synthesis

Lysogeny

• Cascade of transcription activators and anti-terminators

• cI is transcribed at PRE

• Repressor-establishment circuit is turned off• Autogenous repressor-maintenance circuit is

turned on

cII• Establishes cI transcription at PRE

• other functions?– activates transcription from promoter Panti-Q,

located within the Q gene• anti-sense Q-RNA prevents translation of pQ

– Insertion of DNA into the bacterial genome requires the product of int gene

• cII activates transcription of int at Pl

How does the phage enters the lytic cycle?

Cro is the answer!

27.18 The Cro Repressor Is Needed for Lytic Infection

• Cro binds to the same operators as the lambda

repressor protein (cI), but with different affinities.

• Cro has different affinities for each binding site within the

operators

• When Cro binds to OR3, it prevents RNA polymerase

from binding to PRM and blocks the maintenance of

repressor promoter.

Cro

• 9kD subunits

• acts as a dimer

Two effects:

• Prevents repressor transcription via PRM

• Inhibits transcription of early genes from PR an PL

27.18 The Cro Repressor Is Needed for Lytic Infection

• When Cro binds to other operators at OR or OL, it prevents RNA polymerase from expressing immediate early genes, which (indirectly) blocks repressor establishment.

FIGURE 34: The lytic pathway leads to expression

of cro and late genes

Cro represses : PRM, PR & PL

• Binds to OR3

– Blocks transcription at PRM

• Binds to OR2 and OR1 or OL sites

– Inhibits RNA pol binidng– Inhibits the transcription of early genes

• Transcription of late genes occur via pQ

• Lysogeny

– Repressor ( cI) • prevents transcription from PR and PL

• maintains repression via autogenous circuit.

• Lytic Cycle– pN anti-terminator

• allows transcription of early and late genes– Cro

• prevents expression of repressor• prevents transcription of early genes, cII cIII

included

• Lysogeny:

– Interaction of a phage repressor with an operator

– Repressor Binding

• Lytic Cycle:

– Cascade of transcriptional controls

• Transition:

– Relief or establishment of repressor

27.19 What Determines the Balance between Lysogeny and the Lytic Cycle?

• The delayed early stage when both Cro and repressor

are being expressed is common to lysogeny and the lytic

cycle.

• The critical event is whether cII causes sufficient

synthesis of repressor to overcome the action of Cro.

* Repressor determines lysogeny, and

Cro determines the lytic cycle

27.19 What Determines the Balance between Lysogeny and the Lytic Cycle?

FIGURE 35: Repressor determines lysogeny,

and Cro determines the lytic cycle

Lysogeny-Lytic cycle transition

• cII establishes cI expression• cII is unstable

– Susceptible to degradation by host proteases– Mutations in host genes increase lysogeny– hflA and hflB

1. Phages have a lytic life cycle, in which infection of a host

bacterium is followed by production of a large number of phage

particles, lysis of the cell, and release of the viruses.

2. Lytic infection falls typically into three phases. In the first phase

a small number of phage genes are transcribed by the host RNA

polymerase.

3. In phage lambda, the genes are organized into groups whose

expression is controlled by individual regulatory events.

Summary

4. Each operator consists of three binding sites for repressor.

5. The helix-turn-helix motif is used by other DNA-binding

proteins, including lambda Cro, which binds to the same

operators, but has a different affinity for the individual operator

sites, determined by the sequence of helix-3.

6. Establishment of repressor synthesis requires use of the

promoter PRE, which is activated by the product of the cII gene.

Summary

Cascade of gene expression

Groups of genes are turned on ( or off) at particular times.

Stage I: Early genes transcribed by host mRNA poly.

Stage II : Production of enzymes needed for replication.

Stage III : Production of genes for building phage components.

Summary

• The regulator gene that controls the switch from

immediate early to delayed early expression in phage

lambda is identified by mutations in gene N.

• Lambda has two immediate early genes, N and cro,

which are transcribed by host RNA polymerase.

• The N gene is required to express the delayed early

genes.

Summary

• The cro gene code for prevention of expression of cI

• Three of the delayed early genes are regulators.

• The lambda repressor, encoded by the cI gene, is

required to maintain lysogeny.

• Binding to the operator requires the dimeric form so that

two DNA-binding domains can contact the operator

simultaneously.

Summary

• Repressor binding at OR blocks transcription of cro, but

also is required for transcription of cI.

• cII and cIII cause repressor synthesis to be established

and also trigger inhibition of late gene transcription.

• When Cro binds to OR3, it prevents RNA polymerase from

binding to PRM and blocks the maintenance of repressor

promoter.

Summary

• Lysogeny

– Repressor ( cI) • prevents transcription from PR and PL• maintains repression via autogenous circuit.

• Lytic Cycle– pN anti-terminator

• allows transcription of early and late genes– Cro

• prevents expression of repressor• prevents transcription of early genes, cII cIII included

• Lysogeny:– Interaction of a phage repressor with an

operator– Repressor Binding

• Lytic Cycle:– Cascade of transcriptional controls

• Transition:– Relief or establishment of repressor

• The delayed early stage when both Cro and repressor

are being expressed is common to lysogeny and the lytic

cycle.

• The critical event is whether cII causes sufficient

synthesis of repressor to overcome the action of Cro.

* Repressor determines lysogeny, and

Cro determines the lytic cycle

Summary

Video links for Lambda bacteriophage life cycles

http://www.youtube.com/watch?v=lDXA27Zmo-whttp://www.youtube.com/watch?v=3e4BJjbbqBU

http://www.youtube.com/watch?v=_vR-J05mHhQ

Thank you