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Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
Phage stratagies
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Phage stratagies
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Phage stratagies

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Bacterial Phage strategies

Bacterial Phage strategies

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  • 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.
  • 8. Lytic and Lysogenic cycle http://www.youtube.com/watch?v=gU8XeqI7yts
  • 9. 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.
  • 10. 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
  • 11. 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.
  • 12. 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.
  • 13. 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
  • 14. 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.
  • 15. A B Fig A: Early genes switched off, when middle gene are transcribed. Fig B: Early genes are continued to be expressed.
  • 16. 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.
  • 17. 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.
  • 18. 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.
  • 19. 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.
  • 20. • 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.`
  • 21. 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.
  • 22. 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
  • 23. • 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.
  • 24. 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
  • 25. 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)
  • 26. • 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.
  • 27. 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
  • 28. 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.
  • 29. 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.
  • 30. 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
  • 31. 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.
  • 32. • 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.
  • 33. 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
  • 34. 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.
  • 35. 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.
  • 36. cI expression • PRM-promoter region for cI – RM repressor maintenance – Weak promoter
  • 37. 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.
  • 38. 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
  • 39. 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.
  • 40. 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
  • 41. The immunity region of lambda phage Immunity region – OL-cI- OR-cro – Specifies the repressor and the sites to which the repressor acts
  • 42. 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
  • 43. • Lytic Cycle – Cascade of transcriptional controls • Lysogeny – Autogenous circuit – Repressor binding
  • 44. 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.
  • 45. 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.
  • 46. 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
  • 47. • 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
  • 48. 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
  • 49. Repressor: helix-turn-helix motif • Helix 3 • Helix 2 9 aa 7 aa
  • 50. 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
  • 51. Repressor binding to the operator • The repressor binds simetrically to the site • Each N-terminal domain contacts a similar DNA sequence
  • 52. 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
  • 53. 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
  • 54. The operators; three repressor binding sites
  • 55. 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
  • 56. How does the repressor decide where to bind?
  • 57. 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
  • 58. cI mutants show that N-terminal region interacts with RNA polymerase
  • 59. 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
  • 60. 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
  • 61. 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
  • 62. 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.
  • 63. 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
  • 64. 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
  • 65. 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
  • 66. 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.
  • 67. 27.17 Lysogeny Requires Several Events FIGURE 33: The lysogenic pathway leads to repressor synthesis
  • 68. 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
  • 69. 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
  • 70. How does the phage enters the lytic cycle? Cro is the answer!
  • 71. 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.
  • 72. Cro • 9kD subunits • acts as a dimer Two effects: • Prevents repressor transcription via PRM • Inhibits transcription of early genes from PR an PL
  • 73. 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
  • 74. 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
  • 75. • 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
  • 76. • Lysogeny: – Interaction of a phage repressor with an operator – Repressor Binding • Lytic Cycle: – Cascade of transcriptional controls • Transition: – Relief or establishment of repressor
  • 77. 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
  • 78. 27.19 What Determines the Balance between Lysogeny and the Lytic Cycle? FIGURE 35: Repressor determines lysogeny, and Cro determines the lytic cycle
  • 79. 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
  • 80. 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.
  • 81. 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.
  • 82. 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.
  • 83. 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.
  • 84. 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.
  • 85. 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.
  • 86. • 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
  • 87. • Lysogeny: – Interaction of a phage repressor with an operator – Repressor Binding • Lytic Cycle: – Cascade of transcriptional controls • Transition: – Relief or establishment of repressor
  • 88. 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
  • 89. 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
  • 90. Thank you

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