Bacteriophage lambda (l)

1
Bacteriophage lambda (l)

• Transcriptional switches can regulate cellular
  decisions

2
Lysis or Lysogeny

• Lysis Infection by phage produces many progeny
  and breaks open (lyses) the host bacterium
• Lysogeny After infection, the phage DNA
  integrates into the host genome and resides there
  passively
• No progeny
• No lysis of the host
• Can subsequently lyse (lysogeny)
• Bacteriophage lambda can do either.

3
UV Induction
Lysogeny
Lysis
4
Elements of lysogeny

• The phage genome integrated into the host
  bacterial genome is a prophage.
• Bacterium carrying the prophage is a lysogen.
• Lysogens are immune to further infection by
  similar phage because the phage functions are
  repressed in trans.
• Induction of the lysogen leads to excision of the
  prophage, replication of the phage DNA, and lysis
  of the host bacterium.

5
Genes are clustered by function in the lambda
genome
Late control
Virus head tail
Recombination
Control region
Replication
Lysis
cos
origin
promoter
operator
terminator
Not to scale!
6
Immediate early transcription
Transcription by E. coli RNA polymerase initiates
at strong promoters PR , PR, and PL , and
terminates at ts.
6S RNA
7
Antitermination by N protein leads to early gene
expression
gam
int
red
att
xis
N
cI
cro
cII
O
P
Q
S
R
AJ
cIII
Pint
PL
PRM
PR
PRE
PR
tL1
tR1
tR2
t6S
tR3
6S RNA
N protein
Cro
CIII
CII
Q protein
Recombination proteins
Replication proteins
8
Lytic cascade Cro turns off cI, Q protein action
leads to late gene expression
Lytic functions
Replication proteins
Viral head tail proteins
9
Late stage of lytic cascade
High concentrations of Cro turn off PR and PL
. Abundant expression from PR.
Lytic functions
Viral head tail proteins
10
Lysogeny CII and CIII stimulate expression of cI
to make repressor
tint
PRE promoter for repression establishment
Repressor
11
Lysogeny Repressor turns off transcription
PRM promoter for repression maintenance
Repressor
Activated by Repressor binding to oR1 oR2
12
Regulatory mutants of lambda
Clear plaque mutants
Need wild type for lysogeny
Establishment
Maintenance
cI
Yes
Yes
cII
Yes
No
cIII
Yes
No
Act in trans
Virulent mutants (vir)
Act in cis are double mutants in oR /or oL
13
l operators overlap promoters
oR
oR1
oR2
oR3
PR
-10
-35
TTGACT
GATAAT
cro
N
ATAGAT 5
TTAGAT 5
-10
-35
PRM
14
Repressor structure
l repressor is a dimer monomer has 236 amino
acids.
l repressor can bind cooperatively to operator
sub-sites.
15
Use hybrid genes to dissect regulatory schemes

• Place a convenient reporter gene under control of
  the regulatory elements being studied
• Use a known regulatory region to control the
  trans-acting regulatory element

16
l-lac hybrid genes
Place l cI gene under lac control.
Use lacZ as a reporter.
lac p, o
l cI
l pR , OR
lacZ
321
Control amount of l repressor by IPTG.
E. coli with lac repressor, no lacZ.
See effect of l repressor by b-galactosidase
activity
17
l repressor will turn off expression from PR PL
lac p, o
l cI
l pR , OR
lacZ
b-galactosidase
l repressor
IPTG
l repressor acts cooperatively.
18
Repressor stimulates transcription from PRM
lac p, o
l cI
l pRM , OR
lacZ
123
b-galactosidase
l repressor
IPTG
l repressor at oR1 and oR2 stimulates
transcription from pRM.
19
Binding of repressor blocks transcription from pR
but activates pRM
PR
-10
-35
2 dimers of Repressor, bound cooperatively
oR3
RNA Pol
cro
N
oR1
oR2
-10
-35
operator
PRM
promoter
-10
-35
20
Bacteriophage l Events leading to lysis

• lysis or lysogeny (cI or Cro?) ?
• Both lysis and lysogeny
• PR, PL, PR active synthesize N, Cro
• antitermination by N synthesize cIII, cII, Q
• Lysis
• Low Cro binds OR3, shuts off PRM (cI)
• High Cro shuts off PR and PL
• antitermination by Q activation of PR by Cro

21
Bacteriophage l Events leading to lysogeny

• lysis or lysogeny (cI or Cro?) ?
• Lysis and lysogeny
• PR, PL, PR active synthesize N, Cro
• antitermination by N synthesize cIII, cII, Q
• Lysogeny
• cII stimulate expression from PRE (cI repressor)
  and PINT (integrase)
• cIII stabilizes cII
• cI repressor shuts off PR, PL, PR (no lytic
  functions), stimulates PRM

22
Temperate and lytic phage have a different plaque
morphology
Lytic phage clear plaques
23
Induction and immunity of lysogens
l