Bacterial Phage strategies

1. Phage Strategies
Presented by
Amith Reddy
Eastern New Mexico University

2. 27.1 Introduction
What is a bacteriophage ?
A bacteriophage is a virus
that infects and replicates
within bacteria.
Eg: Lambda phage, T4, T7

3. 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).

4. 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.

5. 27.1 Introduction
FIGURE 01: A phage may follow the lytic or lysogenic pathway

6. 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.

7. 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.

9. Lytic and Lysogenic cycle
http://www.youtube.com/watch?v=gU8XeqI7yts

10. 27.2 Lytic Development Is Divided into
Two Periods
•
•
•
•
Ensure replication of DNA
Initiation of replication
Carries DNA polymerase
Engage in Transcription
Result: Phage mRNAs are
transcribed.
Bacterial mRNA replaced by
Phage mRNA.

11. 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

12. 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.

13. 27.3 Lytic Development Is Controlled by a
Cascade
• 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.

14. 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

15. 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.

16. A
B
Fig A: Early genes switched off, when middle gene are transcribed.
Fig B: Early genes are continued to be expressed.

17. 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.

18. 27.4 Two Types of Regulatory Events
Control the Lytic Cascade
• 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.

19. 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.

20. A
B
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.

21. • 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.`

22. 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.

23. 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

24. • 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.

25. 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

26. 27.6 Lambda Immediate Early and Delayed
Early Genes Are Needed for Both Lysogeny
and the Lytic Cycle
• 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. • 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.

28. 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

29. 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.

30. 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.

31. 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

32. 27.7 The Lytic Cycle Depends on
Antitermination by pN
• A group of genes concerned
with regulation are surrounded
by genes needed for
recombination and replication.
• Structural genes are
clustered.
Fig 11: Map of lambda phage, genome is 48,514 bp
* Lytic cycle genes are expressed in polycistronic transcripts from 3 promoters.

33. • 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.
FIGURE 12: Lambda phage genes are
organized in two transcription units.

34. 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.
Fig 13: Lambda has three stages of development

35. Result
• Right end has lysis genes S-R
• Left end has head and tail genes A-J.
•
(6S RNA)
• The late genes form a single
transcription unit starting from PR
•
When pQ is available, 6S RNA is
extended.
(lies bet Q and S)
• Late transcription terminates at site
Transcript length is 194 bases
•
Hence late genes are expressed.

36. 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.

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

38. 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
FIGURE 15: Repressor maintains
lysogeny
proceeding.

39. 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

40. 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.

41. 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

42. The immunity region of lambda phage
Immunity region
– OL-cI- OR-cro
– Specifies the repressor and the sites to which the
repressor acts

43. The immunity region: repressor &
operators
• Virulent mutations
– OL, OR, λ vir
– λ vir
– Grows on lysogens, λ vir mutations allow the
incoming phage to ignore the resident repressor
and enter the lytic cycle

44. • Lytic Cycle
– Cascade of transcriptional controls
• Lysogeny
– Autogenous circuit
– Repressor binding

45. 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 DNAbinding 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.

46. 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.

47. Repressor subunit
DNA-binding Connector Dimerization
N
1
C
92 111 - 113 132
236
Cleavage site
• 27 kb
• Binds to DNA as a dimer
• Uses a helix-turn-helix motif
• Cooperative binding

48. • 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

49. 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
Half site
of 17 bp.
FIGURE 19: The operator is a palindrome

50. Repressor: helix-turn-helix motif
• Helix 3
• Helix 2
9 aa
7 aa

51. 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

52. Repressor binding to the operator
• The repressor binds simetrically to the site
• Each N-terminal domain contacts a similar DNA
sequence

53. 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.
• 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.
Fig 22: Helix-3 determines
DNA-binding specificity

54. λ 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

55. The operators; three repressor binding
sites

56. 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

57. How does the repressor decide where to bind?

58. 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

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

60. 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

61. 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

62. 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

63. 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.

64. 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

65. 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

66. 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

67. 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.

68. 27.17 Lysogeny
Requires Several
Events
FIGURE 33: The lysogenic
pathway leads to repressor
synthesis

69. Lysogeny
• Cascade of transcription activators and antiterminators
• cI is transcribed at PRE
• Repressor-establishment circuit is turned off
• Autogenous repressor-maintenance circuit is
turned on

70. 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

71. How does the phage enters the lytic cycle?
Cro is the answer!

72. 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.

73. Cro
• 9kD subunits
• acts as a dimer
Two effects:
• Prevents repressor transcription via PRM
• Inhibits transcription of early genes from PR an PL

74. 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

75. 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

76. • 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

77. • Lysogeny:
– Interaction of a phage repressor with an operator
– Repressor Binding
• Lytic Cycle:
– Cascade of transcriptional controls
• Transition:
– Relief or establishment of repressor

78. 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

79. 27.19 What Determines the Balance
between Lysogeny and the Lytic Cycle?
FIGURE 35: Repressor
determines lysogeny,
and Cro determines the
lytic cycle

80. 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

81. Summary
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.

82. 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.

83. 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.

84. 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.

85. 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.

86. 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.

87. • 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

88. • Lysogeny:
– Interaction of a phage repressor with an
operator
– Repressor Binding
• Lytic Cycle:
– Cascade of transcriptional controls
• Transition:
– Relief or establishment of repressor

89. Summary
• 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

90. Video links for Lambda bacteriophage life cycles
http://www.youtube.com/watch?v=lDXA27Zmo-w
http://www.youtube.com/watch?v=3e4BJjbbqBU
http://www.youtube.com/watch?v=_vR-J05mHhQ

91. Thank you