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BACTERIOPHAGES

1. BACTERIOPHAGES. PARTICLES WHICH CANNOT GROW BUT ARE REPLICATED BY LIVING HOST CELLS- OBLIGATE INTRACELLULAR PARASITES. 2. BACTERIOPHAGES - LYTIC GROWTH AND LYSOGENY PHAGE STRUCTURE Capsid, Capsomer , chromosome

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BACTERIOPHAGES

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  1. 1 BACTERIOPHAGES PARTICLES WHICH CANNOT GROW BUT ARE REPLICATED BY LIVING HOST CELLS- OBLIGATE INTRACELLULAR PARASITES

  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/PRODUCTION BY HOST 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

  3. 3 PHAGE STRUCTURE fX174 ICOSAHEDRON CHROMOSOME: SINGLE-STRAND CIRCULAR DNA 5,386 NUCLEOTIDES 10 GENES 30 nM TWENTY TRIANGULAR PLATES NUCLEO - CAPSID CAPSOMERS- STRUCTURAL PROTEIN CAPSID TMV - TOBACCO MOSAIC VIRUS CHROMOSOME: SINGLE-STRAND LINEAR RNA ~6,000 NUCLEOTIDES CAPSOMERS INFECTIOUS ONLY RNA AND PROTEIN CRYSTALS

  4. 4 PHAGE T4 CHROMOSOME: DOUBLE STRAND LINEAR DNA ~2 x 105 NUCLEOTIDE PAIRS ~1 x 108 MOLECULAR WEIGHT ~200 GENES HEAD CAPSOMER COLLAR CORE SHEATH BASE PLATE TAIL TAIL FIBER (6) EXTERIOR SPIKES INJECTION - PENETRATION NOBEL HERSHEY RECEPTOR PROTEIN WALL - OUTER MEMBRANE CYTOPLASMIC MEMBRANE CYTOPLASM

  5. 5 T4 GROWTH / PRODUCTION BY HOST CELLS MINUTES AFTER INJECTION

  6. 6 STAGES: 1.ADSORPTION / ATTACHMENT SPECIFICITY-RECEPTORS 2.INJECTION / PENETRATION SHEATH CONTRACTS CHROMOSOME INJECTION SYNTHESIS OF COMPONENTS (REPLICATION) TRANSCRIPTION TRANSLATION ENERGY PRECURSORS RIBOSOMES PRODUCES: VIRAL mRNA VIRAL DNA (RNA) CHROMOSOME VIRAL STRUCTURAL PROTEINS ASSEMBLY / MATURATION DNA PACKAGED TAILS ADDED INTACT VIRUSES PRODUCED LYSIS / RELEASE T4 LYSOZYME PEPTIDOGLYCAN HYDROLYSIS PROVIDED BY HOST SUMMARY: ONE PHAGE-INFECTED CELL PRODUCES ~102 PROGENY IN ONE GROWTH CYCLE BURST SIZE: AVERAGE NUMBER PROGENY / INFECTED CELL

  7. 7 PHAGE PLAQUES ~107 HOST CELLS TOP AGAR AGAR PLATE INCUBATE CONFLUENT GROWTH

  8. 8 PHAGE PLAQUES ~107 HOST CELLS ~10 T4 AND ~107 HOST CELLS TOP AGAR TOP AGAR AGAR PLATE INCUBATE CONFLUENT GROWTH PLAQUES

  9. 9 PLAQUE FORMATION BY LYTIC (VIRULENT) PHAGE FREE PHAGES 30 MIN MANY CYCLES HOST CELLS INFECTED CELL PLAQUE APPEARS CLEAR- HOST CELLS DESTROYED PHAGE INVISIBLE

  10. 10 GROWTH IN LIQUID CULTURE (OF HOST) ~1 x 108 CELLS / ML LOG TURBIDITY TIME

  11. 11 GROWTH IN LIQUID CULTURE (OF HOST) ~1 x 108 CELLS / ML LOG TURBIDITY ADD 2-3 x 108 T4 / ML PHAGE TITER? TIME

  12. 12 • ALL NUCLEIC ACID REPLICATION PROCEEDS THROUGH DOUBLE STRAND FORM • T4 DOUBLE STRAND DNA DOUBLE STRAND PROGENY DNA • MS2 SINGLE STRAND RNA CHROMOSOME + STRAND (ACTS AS mRNA) TRANSLATION YIELDS PHAGE RNA REPLICASE (AND OTHER PROTEINS) + + + + + PROGENY

  13. 13 BACTERIAL VIRUSES COMPARED TO BACTERIA NO RIBOSOMES NO ENERGY GENERATING SYSTEM FEW ENZYMES

  14. 14 TEMPERATE PHAGES – INFECTION IS FOLLOWED BY A. LYTIC GROWTH WITH PRODUCTION OF PROGENY PHAGES AND DESTRUCTION OF THE HOST CELLS OR B. INTEGRATION OF THE PHAGE DNA INTO THE HOST CHROMOSOME AND PASSIVE REPLICATION OF THE PHAGE DNA DURING HOST CHROMOSOME REPLICATON. LYSOGENY

  15. 15 REPRESSOR PROPHAGE BINARY FISSION LYSOGENS; LYSOGENIC; PASSIVE REPLICATION OF PROPHAGE DURING BINARY FISSION OF HOST

  16. 16 PHAGE LAMBDA - l - TEMPERATE LYTIC GROWTH OR LYSOGENY 48,502 BP 30 GENES COS THE l CHROMOSOME COS COHESIVE SITE

  17. 17 LAMBDA GROWTH ADSORPTION - PENETRATION CHROMOSOME CIRCULARIZES LYTIC GROWTH LYSOGENY ~ 50:50 COS COS DNA LIGASE COVALENTLY CLOSED CIRCLE REPLICATION OR LYSOGENY

  18. 18 PHAGE CHROMOSOME SITE HOST CHROMOSOME SITE ATT POP' ATT BOB' 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: + lDNA IS NOW PROPHAGE HOST IS NOW LYSOGEN

  19. 19 l DNA INTEGRATION ATTACHMENT SITE HOST CHROMOSOME REPRESSOR [REPRESSION] GAL= GALACTOSE BIO = BIOTIN OPERON INTEGRASE PROPHAGE LYSOGEN, STABLE, LYSOGENY PASSIVELY REPLICATED

  20. 20 INTEGRATION DETAILS 5' 3' HOST DNA 5' 3' l DNA INTEGRASE CUTS BOTH COMMON CORES REJOINS l AND HOST DNA l PROPHAGE

  21. 21 PROPHAGE INDUCTION DNA DAMAGE REPRESSOR CLEAVAGE LYTIC GENES NO LONGER INHIBITED EXCISION, LYTIC GROWTH, PROGENY, LYSIS ~REPRESSOR FRAGMENT EXCISIONASE REPLICATION

  22. SOS RESPONSE (LYSOGEN) 22 SOS REPRESSOR PHAGE DNA EXCISED; TO BE REPLICATED CELL DIVISION DELAY RecF PROPHAGE & ITS REPRESSOR UV REPAIR TRANS- LESION DNA POLYMERASE RecA (DAMAGE SENSOR) Regulation of the SOS response regulon in E. coli. (A) About 50 genes around the E. coli chromosome are normally repressed by the binding of a LexA dimer (barbell structure) to their operators. Some SOS genes are expressed at low levels, as indicated by single arrows. (B) After DNA damage, the single-stranded DNA (ssDNA) that accumulates in the cell binds to RecA (circled A), forming a RecA nucleoprotein filament, which binds to LexA, causing LexA to cleave itself. The cleaved repressor can no longer bind to the operators of the genes, and the genes are induced as indicated by two arrows. The approximate positions of some of the genes of the SOS regulon are shown.

  23. 23 LYSOGENIC IMMUNITY [l WILL NOT GROW ON A l LYSOGEN] PROPHAGE REPRESSOR l COMES FROM OUTSIDE AND INFECTS NO l REPLICATION

  24. 24 TURBID PLAQUE FORMATION BY TEMPERATE PHAGE FREE PHAGES LYSOGENS 30 MIN HOST CELLS INFECTED CELL MANY CYCLES

  25. 25

  26. 26 MEETING REQUIREMENTS TO BECOME A PROPHAGE – REPRESSOR GENE AND INTEGRASE GENE HAVE UNIQUE PROMOTERS NOT RECOGNIZED BY HOST RNA POLYMERASE WITH GENERAL TRANSCRIPTION SIGMA FACTOR HOST RNA POLYMERASE WITH GENERAL TRANSCRIPTION SIGMA FACTOR TRANSCRIBES FROM PL AND PR PRODUCING TRANSCRIPTION FACTORS WHICH TURN ON REPRESSOR GENE AND INTEGRASE GENE; PRODUCING REPRESSOR & INTEGRASE REPRESSOR INHIBITS TRANSCRIPTION OF ALL GENES INVOLVED IN LYTIC GROWTH AND STIMULATES ITS OWN TRANSCRIPTION INTEGRASE DOES ITS THING – CATALYZES INTEGRATION OF PHAGE DNA INTO HOST CHROMOSOME - NOW A PROPHAGE

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