Transcript Chapter 27 Phage Strategies

Chapter 27
Phage Strategies
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
FIGURE 01: A phage may follow the lytic or lysogenic pathway
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
• 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.
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
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.
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.
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
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
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.
• Three of the delayed early genes are regulators.
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
27.7 The Lytic Cycle Depends on
Antitermination by pN
• 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.
FIGURE 12: Similar controls apply to
left and right transcription
27.7 The Lytic Cycle
Depends on
Antitermination by pN
• Lambda DNA circularizes
after infection; as a result,
the late genes form a single
transcription unit.
FIGURE 13: Lambda has
three stages of development
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.
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.
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 The DNA-Binding Form of the
Lambda Repressor Is a Dimer
• Cleavage of the repressor
between the two domains
reduces the affinity for the
operator and induces a lytic
cycle.
FIGURE 18: Repressor cleavage
induces lytic cycle
27.11 Lambda Repressor Uses a HelixTurn-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
27.11 Lambda Repressor Uses a HelixTurn-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.
FIGURE 22: Helix-3 determines
DNA-binding specificity
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.
FIGURE 25: Lambda repressors
bind DNA cooperatively
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
• 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.
FIGURE 27: Helix-2 interacts
with DNA polymerase
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 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
27.16 A Poor Promoter Requires cII Protein
• PRE has atypical 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.
FIGURE 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
27.18 The Cro Repressor Is Needed for
Lytic Infection
• Cro binds to the same operators as the lambda
repressor, but with different affinities.
• When Cro binds to OR3, it prevents RNA polymerase
from binding to PRM and blocks the maintenance of
repressor promoter.
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
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.
27.19 What Determines the Balance
between Lysogeny and the Lytic Cycle?
FIGURE 35: Repressor
determines lysogeny,
and Cro determines the
lytic cycle