Bacteriophages

BI301

general characteristics of viruses

-virus=poison
-viruses only capable of replicating within living host
-obligate intracellular parasites
-very small
-neither living nor dead: resemble complex toxic molecules (toxic to cell theyre infecting)
-viruses are capable of infecting representatives from all domains of life

structural components of viruses

capsid(protein coat)+nucleic acid(DNA or RNA)=nucleocapsid
-two typess: naked virus and enveloped virus
-simple and complex structures

naked virus

virus without an envelope, capsid only (nucleocapsid)
-simple virus structure

enveloped virus

virus that contains lipid layer surrounding the capsid
-spikes on it which are proteins embedded and recognize specific molecules on host cell surface and bind to host cell receptors
-complex structure

complex virus structure

(bacteriophage): capsid (head), body, tail
-not motile

capsid structure of virus

-capsid proteins form 20 sided geometric shapes
-very stable structure
-the viral genome is packaged inside the capsid
-virus capsids are most often icosahedral

general characteristics of viruses: genetic information

-either DNA or RNA: DNA= circular or linear, double or single stranded
RNA= typically single stranded some ds
-viral genomes are small compared to bacteria

baltimore classification of viruses

7 total; based on the relationship between the viral genomes and how mRNA is synthesized
-Viruses classified by genome structure, polymerase type, envelope, host cells

viruses of bacteria

bacteriophages

bacteriophage facts

-exclusively (only) infect and replicate within bacteria

• most abundant microbe on earth bc it infects most abundant living thing (Prokaryotes)
  -a lot on the planet, wherever bacteria is, bacteriophages are

phage structure

-head (capsid) contains packaged DNA
-tail sheath: can contact and allow DNA to extract into cell, made of viral proteins and attached to head
-plate=made of viral proteins, base of phage
-tail fibers=legs, not motile
-the DNA goes into the host by piercing into the cell of the bacteria

most common phages

T4 and lamda

phage genomes

-T4 is used for phage replication
-genes are arranged by when they are needed
-class 1,2,3
-middle phages=enzymes involved in shutting down host cell and make host a bacteriophage factory

first step of replication of bacteriophages/infection

-adsorption to host cell=attaching virus to cell envelope of bacteria cell
-tail plate binds to cell surface

adsorption in lamda bacteriophage replication

-bacteriophage lamda binds to outer membrane maltose porin on E.coli
-lamda uses this molecule as a signal to attach

flagella attachment for phage adsorption

-bacteriophage can attach to flagellum and wrap its tail around flagella
-flagella must be rotating for infection, phage spins and makes its way to cell surface cause its getting closer

T4 adsorption

-tail pins attach to outer cell wall and tail fibers attach and head empties DNA into membrane

the receptors bacteriophages bind to

LPS, OmpF (porins), TolC (transporters), flagellar motor

bacteriophage replication strategies

lytic cycle and lysogenic cycle
-starts with infection then can either go lytic pathway or lysogenic pathway which eventually leads back to lytic

lytic cycle

-phage hijacks bacterium
-host cell becomes phage factory (basically dead)
-cell lyses, phage releases from cell, results in bacteria cell death (host dies)

replication of lytic bacteriophages steps

1) phage adsorption by diffusing in environment until finds nice receptor to adsorb to
2) penetration: phage DNA is injected into host cytoplasm, host take-over, as soon as phage enters, it is expressed (hijacking step)
3) synthesis: make new phage molecules, biosynthesis of new phage proteins and DNA, destruction of phage chromosome
4) assembly: spontaneous assembly of phage proteins, packaging of phage genome into the heads, holin protein gets inserted into cell membrane which acts as a channel for enzyme endolysin to exit through holins and chew up cell wall and cause cell lysis
5) lysis of host cell, so new particles can release
6) virus' release and go into environment, ends in cell death, increase in bacteriophages

burst size

The number of virus particles released from a lysed host cell. number of new phages produced following replication

timeline of T4 infection

all within 25 minutes

phage replication equals

bacterial death

liquid growth medium with phage replication

-no cells no phage=clear medium
-cells only=cloudy medium
-cells+phage= clear bc entire bacterial population has been lysed

solid medium with phage replication

plaques (holes) are sites of phage replication

lysogenic replication

-lysogeny
-phage DNA integrates into host chromosome
-phage DNA replicates along with host chromosome
-can enter lytic cycle

temperate phages

phages that use both the lytic and lysogenic cycles

replication of bacteriophages: lysogeny steps

1)penetration, one phage DNA (linear genome) in cytoplasm it will circularize
2)phage DNA integrates into bacterial chromosome, brings in new genes

lysogeny: temperate bacteriophages

ex: lamda phage
-phage genome can stably integrated into host chromosome
-prophage and lysogen
-can mold genetics of bacterial populations

prophage

A phage DNA that has been inserted/integrated into a bacterial chromosome.

lysogen

a bacterium/bacterial cell containing a prophage

induction

triggers entry into lytic pathway
-when prophage exits from chromosome and goes to lytic cycle
-if host cell isnt growing well or growing too well, phage causes induction to make more phages
-DNA damage triggers lytic pathway through induction
-ex: UV light or temperature can cause host cell stress and DNA damage so will go through induction to use lytic pathway

insertion of phage DNA into host chromosome

bacterial DNA molecule splits and viral DNA combines and now have spliced viral genome with the prophage

how does it know which pathway to go through for phage replication
using lamda replication

-Cro = when in abundance (increase concentration) will cause lysis pathway
-CII : increase concentration of it will go down lysogenic pathway
-when in lysogeny, increase in C1 occurs which= lamda repressor which causes increase in [Cro] (cause lytic cycle)

lysogenic conversion

-integration of prophage alters host properties
-acquisition of virulence genes
-when a bacterium acquires a new trait from its temperate phage

protection against phages

-restriction/modification systems/genes
-restriction enzymes cleave foreign DNA at specific sequences and creates single stranded sticky ends

restriction gene

encode endonucleases
-recognize specific nucleotide sequence in DNA
-EcoRI is a restriction enzyme and when it finds a sequence, it cuts into backbone and cuts out fragments of DNA and conbines with new DNA and will glue together by DNA ligase which seals up DNA backbone. causes a double strand break

modification systems to protect against phages

-enzymes methylate (add CH3) bases at restriction site and bacterial chromosomes are methylated
-methylation protects DNA from being cut so restriction enzyme cannot cut at that sequence
-restriction enzymes are important tools in molecular biology

transduction

-movement/transfer of genetic material between bacterial cells by temperate phages (can mold genetics of bacterial populations)
-process: host chromosome fragments from bacteria are mixed in with phage DNA and then accidental bacteria DNA fragments get packaged to next host and new host causes a lysogenic conversion as the host genome caned new genetic information

phages can

kill cells and effect the genome

CRISPR- CAS system of phage resistance

-adaptive "immune" response
-defending against foreign DNA (phage or plasmid DNA)
-The bacterium records a virus's DNA (like taking a mugshot).
It saves this record in its own DNA.
If the virus attacks again, the bacterium remembers and uses special scissors (Cas proteins) to cut up the virus before it causes trouble.

CRISPR steps

1. adaptation phase
2. storage
3. expression/processing phase
4. attack phase

adaptation phase of CRISPR

When a phage infects a bacterium, the bacterium can capture a piece of the phage’s DNA.
It inserts this small DNA fragment into a special region of its own genome called CRISPR (which stands for Clustered Regularly Interspaced Short Palindromic Repeats).
These bits of viral (foreign) DNA are called spacers.

storage phase of CRISPR

The CRISPR region becomes a genetic memory bank of past infections.
Each spacer is separated by short, repeated sequences.

expression/processing phase of CRISPR

When the bacterium later encounters a phage (the same or a similar one), it transcribes the CRISPR region into a long RNA molecule.
This RNA is then cut into smaller pieces, called CRISPR RNAs (crRNAs), each containing one spacer (one memory of a virus).

attack phase of CRISPR

The crRNAs guide Cas proteins (CRISPR-associated proteins) to search for matching DNA in an invading phage.
If a match is found (the crRNA matches the phage DNA), the Cas proteins cut and destroy the phage DNA, stopping the infection before it can harm the bacterium.

spacers

foreign DNA sequences that are copied into bacteria chromosome

anatomy of CRISPR locus

1) Leader Sequence:
A short non-coding DNA region located at one end (usually upstream).
It acts like a promoter to start transcription.
It's important for adding new spacers
2) Repeats:
Short identical DNA sequences
That repeat
3) Spacers:
Unique DNA sequences between the repeats.
Each spacer comes from a piece of phage or plasmid DNA captured during an infection.
They serve as a memory of past invaders.
5) Cas Genes (CRISPR-associated genes):
Found near the CRISPR array, often grouped together.
Encode the Cas proteins that perform cutting, processing, and inserting tasks.
Different types of Cas proteins have different roles

an overall model of CRISPR/Cas activity

-spacer acquisition: sequences from invading DNA are incorporated into the CRISPR locus
-crRNA processing: crRNA is transcribed and processed by Cas products and processed into guide RNA's
-effector stage: use immune system against future infection, guide RNA binds and opens up double strand DNA (cleaves) and needs PAM sequence to do so, phage DNA is damaged and destroyed so no more infection

proto spacer adjacent motif

3 base pair sequence adjacent to foreign DNA sequence that will be copied into chromosome so PAM is being scanned to put in memory bank of chromosome
-part of first part of CRISPR activity

purpose of CRISPR

used for gene editing potential
-leads to phage therapy
-In nature, CRISPR protects bacteria and archaea from viruses (phages) and other foreign genetic elements.
It’s a defense system that allows microorganisms to:
"Remember" past infections by storing pieces of viral DNA.
"Recognize" and quickly destroy those invaders if they attack again.

phage therapy

using bacteriophages to treat bacterial infections
-alternative to antibiotics possibly

phage therapy advantages

-exclusively infect bacterial cells not human
-specific to species and strains
-genetically malleable (phages can be bioengineered and change genetic makeup)

phage therapy disadvantages

-lack of controlled studies
-antigenicity: be deemed foreign and cause immune response and can hurt patient (not really an issue)
-bacterial resistance to phages