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BACTERIOPHAGES
PARTICLES WHICH CANNOT GROW BUT ARE REPLICATED
BY LIVING HOST CELLSOBLIGATE 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
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PHAGE STRUCTURE
fX174
CAPSOMERSSTRUCTURAL
PROTEIN
CAPSID
ICOSAHEDRON
CHROMOSOME:
SINGLE-STRAND
CIRCULAR DNA
5,386 NUCLEOTIDES
10 GENES
30 nM
TWENTY TRIANGULAR PLATES
NUCLEO - CAPSID
TMV - TOBACCO MOSAIC VIRUS
CHROMOSOME:
SINGLE-STRAND
LINEAR RNA
~6,000 NUCLEOTIDES
CAPSOMERS
INFECTIOUS
ONLY RNA AND PROTEIN
CRYSTALS
3
4
TAIL
HEAD
PHAGE T4
CHROMOSOME:
DOUBLE STRAND
LINEAR DNA
~2 x 105 NUCLEOTIDE PAIRS
~1 x 108 MOLECULAR WEIGHT
COLLAR
~200 GENES
CAPSOMER
CORE
SHEATH
BASE PLATE
TAIL FIBER (6)
EXTERIOR
SPIKES
NOBEL
HERSHEY
INJECTION PENETRATION
RECEPTOR
PROTEIN
WALL - OUTER MEMBRANE
CYTOPLASMIC MEMBRANE
CYTOPLASM
T4 GROWTH / PRODUCTION BY HOST CELLS
MINUTES AFTER INJECTION
5
STAGES:
6
1.
ADSORPTION / ATTACHMENT
SPECIFICITY-RECEPTORS
2.
INJECTION / PENETRATION
SHEATH CONTRACTS
CHROMOSOME INJECTION
3.
SYNTHESIS OF COMPONENTS (REPLICATION)
TRANSCRIPTION
TRANSLATION
ENERGY
PRECURSORS
RIBOSOMES
PROVIDED BY HOST
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
SUMMARY: ONE PHAGE-INFECTED CELL PRODUCES ~102 PROGENY
IN ONE GROWTH CYCLE
BURST SIZE: AVERAGE NUMBER PROGENY / INFECTED CELL
PHAGE PLAQUES
~107 HOST CELLS
TOP AGAR
AGAR
PLATE
INCUBATE
CONFLUENT
GROWTH
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8
PHAGE PLAQUES
~107 HOST CELLS
TOP AGAR
TOP
AGAR
~10 T4 AND
~107 HOST CELLS
AGAR
PLATE
INCUBATE
CONFLUENT
GROWTH
PLAQUES
PLAQUE FORMATION BY LYTIC (VIRULENT) PHAGE
FREE PHAGES
30 MIN
HOST CELLS
INFECTED CELL
PLAQUE APPEARS
CLEARHOST CELLS
DESTROYED
PHAGE INVISIBLE
MANY
CYCLES
9
10
LOG TURBIDITY
GROWTH IN LIQUID CULTURE (OF HOST)
~1 x 108
CELLS / ML
TIME
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LOG TURBIDITY
GROWTH IN LIQUID CULTURE (OF HOST)
~1 x 108
CELLS / ML
ADD
2-3 x 108
T4 / ML
TIME
PHAGE TITER?
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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
BACTERIAL VIRUSES COMPARED TO BACTERIA
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
NO RIBOSOMES
NO ENERGY GENERATING SYSTEM
FEW ENZYMES
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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?
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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
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REPRESSOR
PROPHAGE
BINARY FISSION
LYSOGENS;
LYSOGENIC;
PASSIVE REPLICATION
OF PROPHAGE DURING
BINARY FISSION OF
HOST
PHAGE LAMBDA - l - TEMPERATE
LYTIC GROWTH OR LYSOGENY
48,502 BP 30 GENES
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THE l CHROMOSOME
COHESIVE SITE
LAMBDA GROWTH
ADSORPTION - PENETRATION
CHROMOSOME CIRCULARIZES
LYTIC GROWTH
LYSOGENY
~ 50:50
COS
COS
DNA LIGASE
COVALENTLY
CLOSED CIRCLE
REPLICATION OR LYSOGENY
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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:
PHAGE
HOST
ATT
ATT
CHROMOSOME
CHROMOSOME
+
POP'
BOB'
SITE
SITE
l DNA IS NOW PROPHAGE
HOST IS NOW LYSOGEN
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l DNA INTEGRATION
ATTACHMENT SITE
HOST CHROMOSOME
REPRESSOR
[REPRESSION]
GAL= GALACTOSE
BIO = BIOTIN OPERON
INTEGRASE
PROPHAGE
LYSOGEN, STABLE, LYSOGENY
PASSIVELY REPLICATED
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INTEGRATION DETAILS
5'
3' HOST DNA
5'
3'
l DNA
INTEGRASE CUTS
BOTH COMMON CORES
REJOINS l AND HOST DNA
l PROPHAGE
PROPHAGE INDUCTION
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DNA DAMAGE REPRESSOR CLEAVAGE
LYTIC GENES NO LONGER INHIBITED
EXCISION, LYTIC GROWTH, PROGENY, LYSIS
~REPRESSOR FRAGMENT
EXCISIONASE
REPLICATION
SOS RESPONSE (LYSOGEN)
SOS
REPRESSOR
CELL DIVISION
DELAY
RecF
PROPHAGE &
ITS REPRESSOR
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PHAGE DNA
EXCISED;
TO BE
REPLICATED
UV
REPAIR
TRANSLESION
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.
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LYSOGENIC IMMUNITY
[l WILL NOT GROW ON A l LYSOGEN]
PROPHAGE
REPRESSOR
l COMES FROM
OUTSIDE AND
INFECTS
NO l REPLICATION
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TURBID PLAQUE FORMATION BY
TEMPERATE PHAGE
FREE PHAGES
LYSOGENS
30 MIN
HOST CELLS
INFECTED CELL
MANY
CYCLES
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MEETING REQUIREMENTS TO BECOME A PROPHAGE –
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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
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