Phage

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Phage

  1. 1. BACTERIOPHAGES 1
  2. 2. BACTERIOPHAGES - LYTIC GROWTH AND LYSOGENY PHAGE STRUCTURE Capsid, Capsomer, chromosome Phage T4 (Head, Collar, Tail, Core, Sheath, Base plate, Spikes, Tail fibers, Specificity, Double -stranded linear chromosome) LYTIC PHAGE GROWTH Attachment (adsorption, specificity) Penetration (injection) Replication - Transcription, translation - Host provides: energy, ribosomes, RNA polymerase. low molecular weight precursors for macromolecular synthesis - Production of viral proteins and nucleic acids Assembly (maturation) (packaging) intact progeny viruses produced Lysis - release of progeny Burst size Plaques (Host, Lawn, Plaques) Phage growth in liquid cultures of host Phages are said to “infect” their host Phage preparations (i.e., suspensions of phages in liquid) are also called “phage lysates” TEMPERATE PHAGES AND LYSOGENY Lambda - Infection : Attachment, Penetration, Circularization of chromosome. Repression of lytic genes, Integration, Attachment site, Lysogeny, Lysogenic immunity, Prophage, Lysogen Prophage Induction Inducing agent Repression abolished, Lytic gene expression. Excision Lytic growth How would you isolate a lysogenic culture if you are given a temperate phage preparation and a culture of host bacteria? 2
  3. 3. 3 PHAGE STRUCTURE CAPSOMERS- STRUCTURAL PROTEIN ICOSAHEDRON CHROMOSOME: SINGLE-STRAND CIRCULAR DNA 5,386 NUCLEOTIDES 10 GENES 30 nM TWENTY TRIANGULAR PLATES NUCLEO - CAPSID CAPSID  X174 TMV - TOBACCO MOSAIC VIRUS CHROMOSOME: SINGLE-STRAND LINEAR RNA ~6,000 NUCLEOTIDES CAPSOMERS INFECTIOUS ONLY RNA AND PROTEIN CRYSTALS
  4. 4. 4 PHAGE T4 HEAD TAIL CAPSOMER CORE SHEATH COLLAR BASE PLATE TAIL FIBER (6) SPIKES CHROMOSOME: DOUBLE STRAND LINEAR DNA ~2 x 10 5 NUCLEOTIDE PAIRS ~1 x 10 8 MOLECULAR WEIGHT ~200 GENES EXTERIOR CYTOPLASM WALL - OUTER MEMBRANE CYTOPLASMIC MEMBRANE RECEPTOR PROTEIN INJECTION - PENETRATION NOBEL HERSHEY
  5. 5. 5 T4 GROWTH MINUTES AFTER INJECTION
  6. 6. 6 <ul><li>1. ADSORPTION / ATTACHMENT </li></ul><ul><li>SPECIFICITY-RECEPTORS </li></ul><ul><li>2. INJECTION / PENETRATION </li></ul><ul><li>SHEATH CONTRACTS </li></ul><ul><li>CHROMOSOME INJECTION </li></ul><ul><li>SYNTHESIS OF COMPONENTS (REPLICATION) </li></ul><ul><li>TRANSCRIPTION </li></ul><ul><li>TRANSLATION </li></ul><ul><li>ENERGY </li></ul><ul><li>PRECURSORS </li></ul><ul><li>RIBOSOMES </li></ul><ul><li>PRODUCES: VIRAL mRNA </li></ul><ul><li>VIRAL DNA (RNA) CHROMOSOME </li></ul><ul><li>VIRAL STRUCTURAL PROTEINS </li></ul><ul><li>ASSEMBLY / MATURATION </li></ul><ul><li>DNA PACKAGED </li></ul><ul><li>TAILS ADDED </li></ul><ul><li>INTACT VIRUSES PRODUCED </li></ul><ul><li>LYSIS / RELEASE </li></ul><ul><li>T4 LYSOZYME </li></ul><ul><li>PEPTIDOGLYCAN HYDROLYSIS </li></ul>PROVIDED BY HOST STAGES: SUMMARY: ONE PHAGE-INFECTED CELL PRODUCES ~10 2 PROGENY IN ONE GROWTH CYCLE BURST SIZE: AVERAGE NUMBER PROGENY / INFECTED CELL
  7. 7. 7 PHAGE PLAQUES ~10 7 HOST CELLS TOP AGAR AGAR PLATE CONFLUENT GROWTH INCUBATE
  8. 8. 8 PHAGE PLAQUES ~10 7 HOST CELLS ~10 T4 AND ~10 7 HOST CELLS TOP AGAR TOP AGAR AGAR PLATE CONFLUENT GROWTH INCUBATE PLAQUES
  9. 9. 9 HOST CELLS INFECTED CELL 30 MIN FREE PHAGES MANY CYCLES PLAQUE APPEARS CLEAR- HOST CELLS DESTROYED PHAGE INVISIBLE PLAQUE FORMATION BY LYTIC (VIRULENT) PHAGE
  10. 10. 10 GROWTH IN LIQUID CULTURE (OF HOST) TIME LOG TURBIDITY ~1 x 10 8 CELLS / ML
  11. 11. 11 TIME LOG TURBIDITY ~1 x 10 8 CELLS / ML ADD 2-3 x 10 8 T4 / ML PHAGE TITER? GROWTH IN LIQUID CULTURE (OF HOST)
  12. 12. <ul><li>ALL NUCLEIC ACID REPLICATION PROCEEDS THROUGH DOUBLE STRAND FORM </li></ul><ul><ul><li>T4 DOUBLE STRAND DNA DOUBLE STRAND PROGENY DNA </li></ul></ul><ul><ul><li>MS2 SINGLE STRAND RNA CHROMOSOME </li></ul></ul>+ STRAND (ACTS AS mRNA) TRANSLATION YIELDS PHAGE RNA REPLICASE (AND OTHER PROTEINS) + + + + + PROGENY 12
  13. 13. 13 BACTERIAL VIRUSES COMPARED TO BACTERIA NO RIBOSOMES NO ENERGY GENERATING SYSTEM FEW ENZYMES VIRULENT - LYSIS OF HOST TEMPERATE - LYSIS / LYSOGENY PHAGES BACTERIA 1. SIZE SMALLER 30 nm - 800 nm LARGER 1000 nm x 3000 nm 2. NUCLEIC ACID CONTENT CHROMOSOME OF DNA OR RNA DNA - CHROMOSOME RNA - mRNA, tRNA, rRNA 3. OUTER STRUCTURES CAPSID - PROTEIN LAYER CELL WALL CYTOPLASMIC MEMBRANE 4. GROWTH ONLY IN LIVING CELLS IN CULTURE MEDIA 5. REPRODUCTION MECHANISM DIRECT SYNTHESIS OF COMPONENTS; ASSEMBLE BINARY FISSION
  14. 14. 14 REPRESSOR PROPHAGE BINARY FISSION LYSOGENS; LYSOGENIC; PASSIVE REPLICATION OF PROPHAGE DURING BINARY FISSION OF HOST
  15. 15. 22 HOST CELLS INFECTED CELL 30 MIN FREE PHAGES LYSOGENS TURBID PLAQUE FORMATION BY TEMPERATE PHAGE MANY CYCLES
  16. 16. 15 PHAGE LAMBDA -  - TEMPERATE LYTIC GROWTH OR LYSOGENY 48,502 BP 30 GENES THE  CHROMOSOME COS COS COHESIVE SITE
  17. 17. 16 LAMBDA GROWTH ADSORPTION - PENETRATION CHROMOSOME CIRCULARIZES LYTIC GROWTH LYSOGENY ~ 50:50 COS COS DNA LIGASE COVALENTLY CLOSED CIRCLE REPLICATION OR LYSOGENY
  18. 18. 17 TRANSCRIPTION OF: REPRESSOR GENE AND EARLY GENES TRANSLATION PRODUCES: REPRESSOR INTEGRASE DNA REPLICATION REPRESSOR: BINDS OPERATORS INHIBITS TRANSCRIPTION OF GENES IN LYTIC GROWTH; STIMULATES OWN TRANSCRIPTION COMPETITION: REPRESSOR AND LYTIC PROTEINS REPRESSOR WINS: SHUTS OFF LYTIC GENES INTEGRATION: SITE SPECIFIC RECOMBINATION BETWEEN: +   DNA IS NOW PROPHAGE HOST IS NOW LYSOGEN ATT POP' ATT BOB' HOST CHROMOSOME SITE PHAGE CHROMOSOME SITE
  19. 19. 18  DNA INTEGRATION ATTACHMENT SITE HOST CHROMOSOME REPRESSOR [REPRESSION] INTEGRASE PROPHAGE LYSOGEN, STABLE, LYSOGENY PASSIVELY REPLICATED GAL= GALACTOSE BIO = BIOTIN OPERON
  20. 20. 19 5' 3' 5' 3' HOST DNA  DNA INTEGRASE CUTS BOTH COMMON CORES REJOINS  AND HOST DNA  PROPHAGE INTEGRATION DETAILS
  21. 21. 20 PROPHAGE INDUCTION DNA DAMAGE REPRESSOR CLEAVAGE LYTIC GENES NO LONGER INHIBITED EXCISION, LYTIC GROWTH, PROGENY, LYSIS ~REPRESSOR FRAGMENT EXCISIONASE REPLICATION
  22. 22. 21 LYSOGENIC IMMUNITY [  WILL NOT GROW ON A  LYSOGEN] PROPHAGE  COMES FROM OUTSIDE AND INFECTS NO  REPLICATION REPRESSOR
  23. 25. 1 A. ESTABLISHING LYSOGENY AFTER INFECTION REQUIRES: c  c  , c  , INT CRO ANTI-c  REPRESSOR (REQUIRED FOR LYTIC GROWTH) B. MAINTAINING LYSOGENY DURING HOST GROWTH REQUIRES: c  LYSIS / LYSOGENY DECISION <ul><li>ESTABLISHMENT </li></ul><ul><li>A. INFECTION - HOST RNA POLYMERASE BINDS P R AND P L ; </li></ul><ul><ul><li>TRANSCRIBES EARLY GENES CRO, c  , c  </li></ul></ul><ul><li>B. c  - STIMULATES RNA POLYMERASE TO TRANSCRIBE FROM P RE PRODUCES c  </li></ul><ul><ul><li>AND FROM P I INTEGRASE </li></ul></ul><ul><ul><li>[BINDS UNIQUE -35 REGIONS] </li></ul></ul><ul><ul><li>[c  PREVENTS c  DEGRADATION] </li></ul></ul>
  24. 26. <ul><li>C. CRO - BINDS O R3 - LIMITS TRANSCRIPTION FROM P RM </li></ul><ul><li>BINDS O R2 O R1 O L3 O L2 O L1 - TURNS OFF EARLY FUNCTIONS </li></ul><ul><li>D. c  - BINDS O R1 > O R2 COOPERATIVELY </li></ul><ul><li>INHIBITS LYTIC GENE EXPRESSION [CRO, c  , OP, etc] </li></ul><ul><li>STIMULATES P RM </li></ul><ul><li>MAINTENANCE </li></ul><ul><li> c  STIMULATES ITS OWN TRANSCRIPTION AND INHIBITS ALL OTHERS </li></ul>2
  25. 27. 3 REGULATORY PROTEINS: c  - REPRESSOR (FACILITATES LYSOGENY) BINDS O R1 > O R2 > O R3 BINDS O L1 > O L2 > O L3 c  - ACTIVATOR OF P RE AND P I c  - PREVENTS c  DEGRADATION CRO - ANTI-c  REPRESSOR (FACILITATES GROWTH) BINDS O R3 > O R2 > O R1 BINDS O L3 > O L2 > O L1 OPERATORS / PROMOTERS: O R /O L - THREE OPERATORS EACH P R AND P RM - OVERLAP O R P L - OVERLAPS O L P RE - PROMOTER FOR REPRESSION ESTABLISHMENT P RM - PROMOTER FOR REPRESSION MAINTENANCE
  26. 28. 4 1.  DNA PENETRATES, CIRCULARIZED, HOST RNA POLYMERASE TRANSCRIBES EARLY FUNCTIONS 2. c  ACTIVATOR STIMULATES TRANSCRIPTION FROM P RE , P I
  27. 29. 5 3. CRO BINDS O R3 ; PREVENTS P RM FROM FUNCTIONING 4. c  REPRESSOR BINDS O R1 O R2 ; SHUTS OFF EARLY GENES TURNS ON P RM O L1 O L2 [CRO / c  RATIO IS CRITICAL]

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