BACTERIOPHAGES 1 PARTICLES WHICH CANNOT GROW BUT ARE REPLICATED BY LIVING HOST CELLS- OBLIGATE INTRACELLULAR PARASITES VIRULENT: DIRECT HOST CELLS TO PRODUCE PROGENY VIRUSES; USUALLY WITH DESTRUCTION/LYSIS OF HOST TEMPERATE: INFECTION LEADS TO PRODUCTION OF PROGENY, OR INTRODUCTION OF PHAGE DNA INTO HOST CHROMOSOME WHERE IT IS REPLICATED PASSIVELY

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 2

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 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 T4 GROWTH / PRODUCTION BY HOST CELLS MINUTES AFTER INJECTION

6 1.ADSORPTION / ATTACHMENT SPECIFICITY-RECEPTORS 2.INJECTION / PENETRATION SHEATH CONTRACTS CHROMOSOME INJECTION 3. SYNTHESIS OF COMPONENTS (REPLICATION) TRANSCRIPTION TRANSLATION ENERGY PRECURSORS RIBOSOMES PRODUCES: VIRAL mRNA VIRAL DNA (RNA) CHROMOSOME VIRAL STRUCTURAL PROTEINS 4. ASSEMBLY / MATURATION DNA PACKAGED TAILS ADDED INTACT VIRUSES PRODUCED 5. LYSIS / RELEASE T4 LYSOZYME PEPTIDOGLYCAN HYDROLYSIS 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 PHAGE PLAQUES ~10 7 HOST CELLS TOP AGAR AGAR PLATE CONFLUENT GROWTH INCUBATE

8 PHAGE PLAQUES ~10 7 HOST CELLS~10 T4 AND ~10 7 HOST CELLS TOP AGAR TOP AGAR PLATE CONFLUENT GROWTH INCUBATE PLAQUES

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 GROWTH IN LIQUID CULTURE (OF HOST) TIME LOG TURBIDITY ~1 x 10 8 CELLS / ML

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)

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 12

13 PHAGESBACTERIA 1. SIZE SMALLER 30 nm 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. GROWTHONLY IN LIVING CELLSIN CULTURE MEDIA 5. REPRODUCTION MECHANISM DIRECT SYNTHESIS OF COMPONENTS; ASSEMBLE BINARY FISSION BACTERIAL VIRUSES COMPARED TO BACTERIA NO RIBOSOMES NO ENERGY GENERATING SYSTEM FEW ENZYMES

TAKE HOME: VIRUSES ARE GROWN (THEY DO NOT GROW) VIRUSES ARE GROWN FROM THEIR GENES BY INFECTED HOST CELLS HOST CELLS EXPRESS VIRAL GENES IN A DEVELOPMENTAL PROCESS PRODUCING VIRAL PROTEINS IN TEMPORAL ORDER IN WHICH THEY ARE NEEDED: ENZYMES FIRST, STRUCTURAL PROTEINS LATER. HOW IS THAT POSSIBLE?

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 14

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

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

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

18 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: ATT POP' ATT BOB' +  DNA IS NOW PROPHAGE HOST IS NOW LYSOGEN HOST CHROMOSOME SITE PHAGE CHROMOSOME SITE

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

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

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

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. SOS RESPONSE (LYSOGEN) RecF SOS REPRESSOR CELL DIVISION DELAY UV REPAIR TRANS- LESION DNA POLYMERASE RecA (DAMAGE SENSOR) PROPHAGE & ITS REPRESSOR PHAGE DNA EXCISED; TO BE REPLICATED 22

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

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

25

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 P L AND P R 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 26

TAKE HOME: (EXAMPLE IS LAMBDA) TEMPERATE PHAGES ENCODE REPRESSOR WHICH PREVENTS LYTIC GROWTH INFECTED HOST CELLS EXPRESS: REPRESSOR AND INTEGRASE FROM PHAGE CHROMOSOME EXPRESSION OF EARLY PHAGE GENES IS DONE BY HOST RNA POLYMERASE WITH GENERAL TRANSCRIPTION SIGMA FACTOR EARLY GENE PRODUCTS INCLUDE TRANSCRIPTION FACTOR WHICH PERMITS REPRESSOR AND INTEGRASE GENE EXPRESSION REPRESSOR IS ALSO ACTIVATOR WHICH STIMULATES ITS OWN GENE EXPRESSION PROPHAGES SHOULD BE INDUCIBLE