Bacteriophage lambda (l)

Transcriptional switches can regulate cellular decisions

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.

UV Induction

Lysis

Lysogeny

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.

Genes are clustered by function in the lambda genome

Late control

Recombination
att int

Control region Replication

cI cro cII O P Q

Virus head Lysis &tail

SR AJ

gam red xis cIII N

Pint

tL1

PL oL PRM PR tR1 PRE tR2 PR t6S cos tR3 oR origin

promoter operator terminator

Not to scale!

Immediate early transcription

Transcription by E. coli RNA polymerase initiates at strong promoters PR , PR, and PL , and terminates at ts.
int gam red xis cIII N

att

cI

cro

cII O P Q

SR

AJ

Pint

tL1

PL oL PRM PR tR1 PRE oR

tR2 PR t6S tR3

6S RNA N Cro

Antitermination by N protein leads to early gene expression

N
att

N
cI cro

N
cII O P Q SR AJ

int

gam red xis cIII N

Pint

tL1

PL

PRM PR tR1 PRE

tR2 PR t6S tR3

6S RNA

N protein
CIII Recombination proteins

Cro
CII Q protein

Replication proteins

Lytic cascade: Cro turns off cI, Q protein action leads to late gene expression
Cro
int gam red xis cIII N

Cro

Q
cII O P Q SR AJ

att

cI

cro

Pint

tL1

PL oL PRM PR tR1 PRE oR

tR2 PR t6S tR3

Lytic functions Replication proteins Viral head & tail proteins

Late stage of lytic cascade

High concentrations of Cro turn off PR and PL . Abundant expression from PR. Cro
att int gam red xis cIII N

Cro
cI cro cII O P Q

Q
SR AJ

Pint

tL1

PL oL PRM PR tR1 PRE oR

tR2 PR t6S tR3

Lytic functions Viral head & tail proteins

Lysogeny: CII and CIII stimulate expression of cI to make repressor

CII

CIII

CII

att

int

gam red xis cIII N

cI

cro

cII O P Q

SR

AJ

tint Pint

tL1

PL oL PRM PR tR1 PRE oR

tR2 PR t6S tR3 PRE = promoter for repression establishment

Int

CI

Repressor

Lysogeny: Repressor turns off transcription

CI
gam red xis cIII N

CI

att

int

cI

cro

cII O P Q

SR

AJ

Pint

tL1

PL oL PRM PR tR1 PRE oR

tR2 PR t6S tR3

PRM = promoter for repression maintenance Activated by Repressor binding to oR1 & oR2

CI

Repressor

Regulatory mutants of lambda

Clear plaque mutants

cI cII cIII

Need wild type for lysogeny: Establishment Maintenance Yes Yes

Yes Yes No No

Act in trans Virulent mutants (vir) Act in cis : are double mutants in oR &/or oL

l operators overlap promoters

oR :
oR3 oR2
-35 TTGACT

oR1

PR
-10 GATAAT

N
TTAGAT 5 -10 ATAGAT 5 -35

cro

PRM

Repressor structure
l repressor is a dimer; monomer has 236 amino acids.
C-te rminal domain: prote in-prote in inte r action; dime rization and coope r ativ ity Conne ctor N-te rminus: DNA binding; He lix-Turn-He lix motif

ope rator

l repressor can bind cooperatively to operator sub-sites.

ope rator oR2

ope rator oR1

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

l-lac hybrid genes

Place l cI gene under lac control. lac p, o l cI Use lacZ as a reporter. l pR , OR 321 lacZ

Control amount of l repressor by [IPTG].

E. coli with lac repressor, no lacZ.

See effect of l repressor by b-galactosidase activity

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.

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.

Binding of repressor blocks transcription from pR but activates pRM

PR
-35 -10

oR3

2 dimers of Repressor, bound cooperatively

cro
RNA Pol
-10 -35

oR2

oR1

PRM
-35

= operator
-10 = promoter

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

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

Temperate and lytic phage have a different plaque morphology

Temperate phage generate turbid plaques Mutants of phage that have lost the capacity to lysogenize form clear plaques

lysoge nize d ce lls lyse d ce lls lyse d ce lls

uninfe cte d ce lls

Lytic phage: clear plaques

Induction and immunity of lysogens

l

A l lysogen Spontaneously, 1/1000 lysogens will induc e, i.e. th e l l prophage will excise , replicate and lyse the cell. + UV tre atment leads to induc tion of virtually all lysogens in a culture. Lyso gens areimmune to further infectio n with similar (lambdoid) phage