BACTERIOPHAGES

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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 TOPRODUCTION OF PROGENY,

ORINTRODUCTION 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 producedLysis - release of progeny

Burst sizePlaques (Host, Lawn, Plaques)Phage growth in liquid cultures of hostPhages are said to “infect” their hostPhage 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 InductionInducing agent Repression abolished, Lytic gene expression.ExcisionLytic growth

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3PHAGE STRUCTURE

CAPSOMERS-STRUCTURAL PROTEIN

ICOSAHEDRONCHROMOSOME:

SINGLE-STRANDCIRCULAR DNA5,386 NUCLEOTIDES10 GENES

30 nMTWENTY TRIANGULAR PLATESNUCLEO - CAPSID

CAPSID

fX174

TMV - TOBACCO MOSAIC VIRUS

CHROMOSOME:SINGLE-STRANDLINEAR RNA~6,000 NUCLEOTIDES

CAPSOMERSINFECTIOUSONLY RNA AND PROTEINCRYSTALS

4PHAGE T4H

EA

DTA

IL

CAPSOMER

CORE

SHEATH

COLLAR

BASE PLATE

TAIL FIBER (6)

SPIKES

CHROMOSOME:DOUBLE STRANDLINEAR DNA~2 x 105 NUCLEOTIDE

PAIRS~1 x 108 MOLECULAR

WEIGHT~200 GENES

EXTERIOR

CYTOPLASM

WALL - OUTER MEMBRANECYTOPLASMIC MEMBRANE

RECEPTORPROTEIN

INJECTION - PENETRATIONNOBEL

HERSHEY

5T4 GROWTH / PRODUCTION BY HOST CELLS

MINUTES AFTER INJECTION

61. ADSORPTION / ATTACHMENT

SPECIFICITY-RECEPTORS

2. INJECTION / PENETRATIONSHEATH CONTRACTS CHROMOSOME INJECTION

3. SYNTHESIS OF COMPONENTS (REPLICATION)

TRANSCRIPTIONTRANSLATIONENERGYPRECURSORSRIBOSOMES

PRODUCES: VIRAL mRNAVIRAL DNA (RNA) CHROMOSOMEVIRAL STRUCTURAL PROTEINS

4. ASSEMBLY / MATURATIONDNA PACKAGEDTAILS ADDEDINTACT VIRUSES PRODUCED

5. LYSIS / RELEASET4 LYSOZYMEPEPTIDOGLYCAN HYDROLYSIS

PROVIDED BY HOST

STAGES:

SUMMARY: ONE PHAGE-INFECTED CELL PRODUCES ~102 PROGENY IN ONE GROWTH CYCLE

BURST SIZE: AVERAGE NUMBER PROGENY / INFECTED CELL

7PHAGE PLAQUES

~107 HOST CELLS

TOP AGAR

AGARPLATE

CONFLUENTGROWTH

INCUBATE

8PHAGE PLAQUES

~107 HOST CELLS ~10 T4 AND~107 HOST CELLS

TOP AGARTOP

AGAR

AGARPLATE

CONFLUENTGROWTH

INCUBATE

PLAQUES

9

HOST CELLSINFECTED CELL

30 MIN

FREE PHAGES

MANYCYCLES

PLAQUE APPEARS CLEAR-

HOST CELLS DESTROYED

PHAGE INVISIBLE

PLAQUE FORMATION BY LYTIC (VIRULENT) PHAGE

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GROWTH IN LIQUID CULTURE (OF HOST)

TIME

LO

G T

UR

BID

ITY ~1 x 108

CELLS / ML

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TIME

LO

G T

UR

BID

ITY ~1 x 108

CELLS / ML

ADD2-3 x 108

T4 / MLPHAGE 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

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PHAGES BACTERIA

1. SIZESMALLER

30 nm - 800 nmLARGER

1000 nm x 3000 nm

2. NUCLEIC ACID CONTENT

CHROMOSOME OF DNA OR RNA

DNA - CHROMOSOMERNA - mRNA, tRNA,

rRNA

3. OUTER STRUCTURES

CAPSID - PROTEIN LAYER

CELL WALLCYTOPLASMIC

MEMBRANE

4. GROWTH ONLY IN LIVING CELLS IN CULTURE MEDIA

5. REPRODUCTION MECHANISM

DIRECT SYNTHESIS OF COMPONENTS;

ASSEMBLEBINARY FISSION

BACTERIAL VIRUSES COMPARED TO BACTERIA

NO RIBOSOMESNO ENERGY GENERATING SYSTEMFEW ENZYMES

TAKE HOME:VIRUSES ARE GROWN (THEY DO NOT GROW)

VIRUSES ARE GROWN FROM THEIR GENES BYINFECTED HOST CELLS

HOST CELLS EXPRESS VIRAL GENES IN A DEVELOPMENTAL PROCESS PRODUCINGVIRAL 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 PROGENYPHAGES 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 FISSIONLYSOGENS;LYSOGENIC;PASSIVE REPLICATION

OF PROPHAGE DURINGBINARY FISSION OFHOST

16PHAGE LAMBDA - l - TEMPERATE

LYTIC GROWTH OR LYSOGENY48,502 BP 30 GENES

THE l CHROMOSOME

COS

COS

COHESIVE SITE

17LAMBDA GROWTHADSORPTION - PENETRATION CHROMOSOME CIRCULARIZES

LYTIC GROWTHLYSOGENY

~ 50:50

COSCOS

DNA LIGASE

COVALENTLYCLOSED CIRCLE

REPLICATION OR LYSOGENY

18TRANSCRIPTION 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:

ATTPOP'

ATTBOB'

+

l DNA IS NOW PROPHAGEHOST IS NOW LYSOGEN

HOSTCHROMOSOME

SITE

PHAGECHROMOSOME

SITE

19 l DNA INTEGRATION

ATTACHMENT SITE

HOST CHROMOSOME

REPRESSOR[REPRESSION]

INTEGRASE

PROPHAGE

LYSOGEN, STABLE, LYSOGENYPASSIVELY REPLICATED

GAL= GALACTOSEBIO = BIOTIN OPERON

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5' 3'

5' 3'

HOST DNA

l DNA

INTEGRASE CUTSBOTH COMMON CORES

REJOINS l AND HOST DNA

l PROPHAGE

INTEGRATION DETAILS

21PROPHAGE 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

SOSREPRESSOR

CELL DIVISIONDELAY

UVREPAIR

TRANS-LESION

DNAPOLYMERASE

RecA(DAMAGESENSOR)

PROPHAGE &ITS REPRESSOR

PHAGE DNAEXCISED;

TO BEREPLICATED

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23LYSOGENIC IMMUNITY[ l WILL NOT GROW ON A l LYSOGEN]

PROPHAGE

l COMES FROMOUTSIDE AND

INFECTS

NO l REPLICATION

REPRESSOR

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HOST CELLSINFECTED CELL

30 MIN

FREE PHAGES LYSOGENS

TURBID PLAQUE FORMATION BYTEMPERATE PHAGE

MANYCYCLES

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MEETING REQUIREMENTS TO BECOME A PROPHAGE –

REPRESSOR GENE AND INTEGRASE GENE HAVE UNIQUEPROMOTERS NOT RECOGNIZED BY HOST RNA POLYMERASEWITH GENERAL TRANSCRIPTION SIGMA FACTOR

HOST RNA POLYMERASE WITH GENERAL TRANSCRIPTIONSIGMA FACTOR TRANSCRIBES FROM PL AND PR PRODUCINGTRANSCRIPTION FACTORS WHICH TURN ON REPRESSORGENE AND INTEGRASE GENE; PRODUCING REPRESSOR &INTEGRASE

REPRESSOR INHIBITS TRANSCRIPTION OF ALL GENES INVOLVED IN LYTIC GROWTH AND STIMULATES ITS OWNTRANSCRIPTION

INTEGRASE DOES ITS THING – CATALYZES INTEGRATION OFPHAGE DNA INTO HOST CHROMOSOME - NOW A PROPHAGE

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TAKE HOME: (EXAMPLE IS LAMBDA)TEMPERATE PHAGES ENCODE REPRESSOR WHICHPREVENTS 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