Bacteriophage Genetics

Virus/phage: DNA or RNA, ds or ss

Intracellular Extracellular
! Replicate in host ! “virion”

! Produce new viruses ! Metabolically inert

Icosahedral:
! !, T4, T2

Filamentous
! M13

Heterologous expression in a different species of Sulfolobus

Heterologous expression in E. coli

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 Phage lytic life cycle
Stages of the lytic life cycle
1. Adsorption (attachment)
• Specific surface receptor
• Pili (M13)
• Transport protein, e.g. MBP (!)

Mutations: very useful for determining gene function

Mutations: provide insight into natural adaptive
mechanisms, e.g. HIV, SARS

http://www-db.embl-heidelberg.de/jss/servlet/de.embl.bk.wwwTools.GroupLeftEMBL/ExternalInfo/fuller/icos0.html

! D- mutant (lacking protein D)

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Stages of the lytic life cycle
Stages of the lytic life cycle
2. Penetration
3. Replication of viral nucleic acid
• T4: lysozyme-like holin secreted from
phage • Early phage genes expressed and
host enzymes sequestered
• Small hole
• Contract tail sheath 4. Synthesis of coat proteins
• DNA injected • Late phage genes expressed

http://www-micro.msb.le.ac.uk/224/Phages.html

How did the phage produce holin?

Stages of the lytic life cycle Stages of the lytic life cycle
5. Assembly 6. Phage release
• Structural proteins (heads, tails) • Holin synthesised
• Catalytic proteins • Lysis
• Assemble structural proteins • Most phage burst cells
• Maturation process (DNA into • M13 causes outfolding (extrusion)
phage heads) and does not puncture the cell
• Icosahedral:
I h d l aggregation i structurall • Burst size: typically 50-100 pfu/cell
proteins, condensation DNA into • Lysate: titer of lysate, e.g. 1011 pfu/ml
heads, attachment of tail
• Multiplicity of infection (MOI): number of
• Filamentous: one step process absorbed phage per cell

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 Phage lytic life cycle Phage life cycle: 2 distinct phases
Lytic Lysogenic
• Phage factory • Stable replication with
• Most
M t phages
h host (not a factory)
kill the host • Type 1 (!): integrated
• Virulent: lytic Type 2 (P1): circular, not
cycle only integrated, like a plasmid
• Plaques: clear • Temperate: lytic &
exceptt phage
h lysogenic
like M13 • Only dsDNA phages
• Plaques: turbid

Some important terms:

! integration (prophage insertion)
• Temperate phage • DNA injected (linear)
• Lysogen • DNA must circularise (via cos sites)
• Integration • Brief period of gene expression
• Repressor protein stops lytic cycle
• Induction: from stable lysogen to lytic cycle
• Site-specific recombination occurs
• Prophage: “before phage” – therefore means between ! and chromosome
lysogen – relates to history of discovery – use
as in, “prophage induction”
• Immunity: resistance to reinfection by same
or like-phage

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 ! circularisation ! integration
COS
5’
COS 3’ CCCGCCGCTGGA

GGGCGGCGACCT 3’
5’
Site-specific
Site-
recombination
circularise

Integrase
! attachment site ((att
att))

!: Overview of gene expression

Lytic and lysogenic pathways both require
• Orderly series of gene expression events immediate early gene expression
during the lytic cycle
• Early genes: DNA replication L ti cycle
Lytic l then
th requires
i l t gene expression
late i
• Late genes: heads, tails, lysis
• Gene organisation reflects the sequence Lysogeny requires expression of the CI repressor
of events needed in the lytic cycle
• Genes are arranged in operons
• Only ~8 transcripts
• Limited number of key genetic control
points

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T4

http://www.blc.arizona.edu/marty/411/Modules/Lectures/Figures/lambda_map.GIF

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 Genetic map of phage !

Recombination Immunity late

Head
Tail
early

Replication Lysis
PL
LEFT
RIGHT
PR

cos att cos

Antiterminators:
• Enable transcription to proceed through
regulatory regions that would otherwise cause
termination
• Convert RNAP into non-terminating g form
• Antiterminator acts on promoter region or
termination region

transcription
transcription terminator
start site
promoter structural gene
region

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 Gene expression in the ! lytic cycle
• Requires O,P

• When ! infects a new host, can ! gene

expression commence?

TL1 PL PR TR1
antiterminator N CRO
N N
nutLPL PR nutR TR3 C D

Lysogeny pathway DNA replication O,P Q

PR’ (PR2) TR4

qut
Q
PR’ (PR2) late genes
Lytic pathway

Gene expression in the ! lysogenic cycle

Gene expression in the ! lytic cycle
• Lytic cycle needs to be repressed
• Synthesis of late gene products occurs
• CI is required
from one long transcript originating from
PR’ • CI is expressed from PRM (maintenance)

• It literally
lit ll ttakes
k minutes
i t ffor sequential
ti l
translation of the mRNA to occur PR e.g. O,P

• In contrast, synthesis of DNA occurs

PL PRM
immediately e.g. int CI
• This allows DNA to be available for
CI
packaging once heads and tails are PR
available X
X PL PRM
CI
CI CI "

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 Where did CI initially come from?
Gene expression in the ! lysogenic cycle CI: repressor PL, PR & activator PRM
• Phage enters cell
CII: activator CI gene expression
• Expression from PL, PR
CIII: stabiliser of CII protein (CII is labile)
• Integrase (Int) expressed early
After initial infection by !
• ! integrates
• CIII expressed from PL

• CII expressed from PR

• CI inhibits PL, PR
• CIII stabilises CII
• ! stably integrated and maintained • CII activates PRE (establishment)

• CI expressed from PRM PRE

• But, CI required for PRM activity • Promoter for CI
• Where did CI come from in the 1st place?! • 10-fold higher expression than PRM
• Provides initial high level burst of CI

Establishing and maintaining CI

PR CII
• Initial PL, PR expression # CIII, CII
CIII PL • CIII/CII activate PRE
• PRE # CI
• CI represses PL, PR
PR
• No more CIII, CII & PRE expression
PL PRM PRE
• CI maintained by PRM
CI

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One more role for CII
• CII activates expression of PI Phage immunity
• PI produces integrase In a ! lysogen, CI prevents any ! (or !-like)
phage from infecting
• Q
Q: How does this occur?

!CI mutants
• !CI : defective CI
-

• !CIvir : defective operator where CI binds

Q: Will !CI- be constitutively lytic or lysogenic?

Q: Will !CIvir be constitutively lytic or lysogenic?
Q: Will the effect be the same if !CIvir or !CI-
enters a ! lysogen?

The decision between lytic and lysogenic

CI and CRO have differential affinity for OL
and OR (operator sequences of PL and PR)
CI affinity: OL1 > OL2 > OL3; OR1 > OR2 > OR3

[CI] Binding sites Action

OL1 Repress PL PR
Low OR1 Activate PRM

OL1 OL2 Complete

C l t repression
i PL PR
Medium OR1 OR2 Activate PRM
OL1 OL2 OL3 Complete repression PL PR
High OR1 OR2 OR3 Repression PRM

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 CRO affinity: OL3 > OL2 > OL1; OR3 > OR2 > OR1
CI synthesis is controlled by autoregulation
• CI activates its own expression
• When [[CI]] is high,
g CI represses
p its own
expression
• When [CI] falls, CI again activates its own
expression
• The ability to both activate and repress its
own expression i is
i termed
t d autoregulation
t l ti CRO synthesis
is controlled by
autoregulation

CI and CRO are mututally exclusive CII: the critical determinant

• Cellular levels controlled by:
PR CII CRO
• Synthesis
CIII PL
CI inhibits CRO • Stability (labile)
CRO inhibits CI CIII senses MOI: high MOI # lysogeny
CII/CIII activates CI CII cleaved by HflA & HflB (host proteases)
CRO PR
HflAB levels controlled by physiology
PL CI PRM PRE
Q: Would a hflAB mutant be constitutively lytic or
lysogenic?

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 Mutants
Revertant: mutation reversal to wild-type Understanding ! Replication: use of mutants
Suppressor mutation (2nd site revertant): • Gro mutants: E. coli mutants that do not
compensatory mutation that alleviates the support ! replication
effects of another mutation
• Mutated
M E colili # strains
d E. hi h ! could
i iin which ld

C
Wild-type Mutant Revertant Suppressor
not replicate
- GLU(-) LYS(+) GLU(-) LYS(+) • Mutations in: dnaJ, dnaP (DNA polymerase)
• Mutate !: !O, !P
+ ARG(+)
( ) ARG(+)
( ) ARG(+)
( ) ASP(-)
()
Intramolecular (cis) suppressor

Intermolecular (trans)
* * suppressor

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 Understanding ! Replication: use of mutants Understanding ! Replication: use of mutants
• Revertants: • Revertants:
• Mutate !O, !P and look for revertants • E. coli Gro mutants to reestablish replication
(reestablishing replication) of !O or !P mutants
• Intramolecular • Gro mutants not successful with !O
• mutation reversal • Gro mutants (dnaB) successful with !P
• 2nd site
• Intermolecular
• Mutations in !O compensates for !P (and Q: what can you infer about the interactions of
vice versa) !O, !P and DnaB?
• Therefore, O and P interact

Understanding ! Replication: use of mutants Understanding ! assembly: use of mutants

• ! “tiny” (ti) plaque mutants • ! and E. coli proteins required
• Reduced burst size • Mutants at numerous stages of assembly
• E. coli coinfected with ! wild-type
yp and ti,, onlyy • !E mutation suppressed
pp in GroE
obtain normal size plaques
• ti mutation: oriT • !E $ GroE (GroEL/GroES)
• Wild-type oriT sequesters DNA replication • Discovery of the main protein folding
machinery machine in E. coli

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 ! recombination
! Rolling circle replication • ! recombination occurs in an E. coli recA strain
• ! DNA is packaged in linear form • Therefore .
• Terminase (Ter) excises at 2 cos sites, and • ! mutants: red locus (recombination deficient)
packages a single copy of !
• bet,
bet exo (exonuclease)
cos cos cos
• Reduced burst size
• Recombination is part of the normal ! life-cycle
cos
Ter cos cos
cos

Ter
cos cos Ter
Red
+ (dimerisation) cos

! recombination ! recombination
• E. coli recombination machinery plays a role: • ! red, gam: burst size ~4 pfu/cell
• ! gam # Gam # inactivates E. coli RecBCD • Continued growth and passaging of the phage
• RecBCD inactivates rolling circle replication led to large plaques
• ! mutation: chi
• ! red, gam: few phage only • chi promotes RecA activity and compensates
• E. coli recA ! red, gam: no phage for ! red, gam
• Therefore, RecA .
Q: E. coli recA,, recBCD ! red,, g
Q gam: p
phage?
g In E. coli a chi sequence is present every ~4kb
4kb

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 Prophage induction
• DNA damage causes induction (e.g. UV)
• SOS response
• RecA # RecA* (protease activity)
• RecA*
R A* cleaves
l L
LexA
A (repressor
( off SOS)
• RecA* cleaves CI
• Damaged cell becomes lytic (rats deserting a
sinking ship)

Factors controlling lysis vs lysogeny:

• RecA* (CI)
• MOI (CIII)
• HflA/B (CII)

Prophage induction: unravelling the

mechanism
• Mutation studies identified int (integrase)
• How involved in excision?
• Why not excision after integration?

Prophage curing
• !CI857: temperature sensitive (TS) mutant
• !CI857 at 42ºC # clear plaques
• !CI857 at 30ºC # turbid plaques
CI857 = CIts
Q: At which temperature is CI857 functional?

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Prophage curing
• E. coli !CI857 at 42ºC/45min # 30ºC (lytic)
• E. coli !CI857 at 42ºC/5min # 30ºC (many
nonlysogenic bacteria)
• prophage curing: excision of ! at 42ºC, but
repression of lysis at 30ºC
Q: How are int
and xis
• Mutate E. coli !CI857 regulated to
• Look for mutated strains that do not support control
prophage curing integration
• Identified: int and xis (excisionase) and
excision?

Retroregulation • PI is within xis

Regulation by sequences downstream of the • The transcript from PI terminates before the
gene RNase III site
• After infection, int and xis expressed from PL • Therefore, Int, but not Xis, is produced from PI
• However,, RNase cleaves at RNase III site
downstream of int/xis genes
• Therefore little Int or Xis produced

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• After induction, int and xis are both stably
expressed from PL because there is no RNase
III site
• This is because, the integration of ! at the att
site,, p
places the RNase III site far away
y from
the end of the PL transcript
• Int has specificity for the attP (phage) and attB
(bacterium) sites
• Xis has specificity for the hybrid attP/B sites
• Therefore:
• Int can not cause excision on its own
• Excision can only occur after induction (when
Xis is synthesised)

POB’

BOP’

http://www-micro.msb.le.ac.uk/224/Phages.html

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