18- Bacteriophages

Bacteriophages

Viruses that infect bacterias. They cannot infect us.

Phage = eat in latin

Prokaryotic viruses

• must bind to host cell receptor

• must cross a cell wall

  • gram^- hosts also cross two membranes

  • must not damage host initially

Prokaryotic virus

• use host nucleotides, amino acids, ATP

  • replicate viral genome, build capsid, build new viruses

• Exit through cell wall

  • usually lyse host cells

Bacteriophage Life Cycle

• Attachment to host cell proteins

  • Normal bacterial cell surface receptors (sugar uptake, signaling, conjugation)

  • viruses takes + uses host proteins

• Injects genome through cell wall into cytoplasm

  • like shooting bacteria

Lytic Cycle

bacteriophage quickly replicates, killing host cell. It explodes, gets out fast, T4

Lysogenic cycle

• Bacteriophage is dormant

• Integrates into cell chromosome as a prophase

• can reactivate and become lytic

• less violent, chills after inserting DNA, lambda

The Lytic Phase (life cycle)

• Use cell components to synthesize capsids

• assemble progeny phases

• exit from cell

The Lytic Phase, Lysis

• Makes protein to break peptidoglycan

• Bursts host cell to release progeny phase

The Lytic Phase, Slow release

Filamentous phages can slip individual progeny out through cell envelope

Coliphages

Viruses that infect E. coli (T4 and lambda)

T4 and lambda

double-stranded linear DNA genome

T4 phage

• lytic

• capsid head, tail

• virulent lytic (intemperate)

Lambda phage

• lysogenic (temperate)

• capsid head, tail

Bacteriophage T4

• complicated structures

• 170 genes

• 10 capsid proteins

• tail fibers bind the host

• receptor = OmpC porin, outer membrane protein

• long tail injects DNA

Absorption and DNA injection

DNA is injected through outer membrane, cell wall, inner membrane, and finally into the cytoplasm (in gram negaice)

Phage T4 genome replication

• upon entry, a phage genome forms a circle

• early genes transcribed

  • take control of the cell, destroy chromosome

• use cell nucleotides to replicate genome

• “rolling circle replication”

• continuous replication of many copies of the genome. Replicates as it rolls

Phage T4 genome replication

• progeny genomes are linked in a concatemer (several genomes linked together)

• cut with an offset, so that individual linear genomes have slight overlaps

Packaging of T4 DNA into virus heads

• genome is packaged slightly longer than the complete phage chromosome (103%)

• Sequence begins and ends at different points in different virions

• start and end are not static, order does not matter

Phage Particles Self-Assemble

Late genes transcribed

• capsid particles”

• head polymerizes around progeny DNA

• Tail fibers, long tail made

• head, tail, tail. fibers assemble

• Lysis protein made

  • destroys cell wall, releases progeny

Lytic cycle of T4 phage

1. DNA injection

2. degrades host DNA

3. mRNA of phage made and replicated

4. heads and tails made

5. heads filled with DNA

6. virions form

7. host cell lysis

Lysogenic Bacteriophage

Some phages maintain a stable relationship with the host cell where they don’t kill and stay with the host: temperate phages

Temperate phages are capable of lysogeny

• Integrate viral genome (prophage) into host DNA (a bacterium harboring a prophage is called lysogen)

• prophage DNA is mostly dormant

• doesn’t enter the lytic cycle until induced (DNA damage/ very bad environment)

• only a single lysogenic virus of a particular type can be present in a host cell

temperate phages

integrate into host DNA. replicated bc the virus is part of the chromosome

lysogen

a bacterium harboring a prohage

Temperate coliphage lambda

capsid head, tail

• double stranded DNA genome (50 genes)

• linear DNA with cohesive sends (cos sites)

• DNA circularizes inside the host

• lambda phase receptor in e.coli is porin

Lambda phage DNA

• ds linear in phage head, circular in host

• tandem repeating ends combine in host to give a circular configuration at the ligated cos sire

• cos recognition site determine where the genome is cut and packaged

Replication of lambda genome

Theta replication (theta; circle-to-circle)

• at the beginning

• initiates at the ori site

• bi-directional replication

Rolling circle replication

• continuous replication of many copies of the genome

• rolls in 2 directions always

• long chains of concatenated genomes

• cutting occurs at cos sites to generate linear form for packaging

temperate

lambda

Lambda

• cuts a cos sites

• lysogenic, lytic when induces

T4

• always lytic

• cuts according to DNA lengths (103% of genome size)

Transduced cell

contains host DNA and phage DNA

Specialized Transduction

• Some viruses can integrate their viral genome into the bacterial chromosome (lysogeny) at att sites

• when entering lytic cycle, bacterial genes adjacent to the viral attachment (att) sites are sometimes picked

lytic transduction

• in the induction of the lytic cycle sometimes prophage is incorrectly excised from bacterial chromosome

• phage DNA incorporates some bacterial genes

• when the bacterial cell is lysed, phages are released including the host DNA

• the new host acquires both phage DNA and genes from the previous host

Slow-release replication

• some phages can push individual through the cell envelope

• host cells grow slowly but don’t die

Culturing Bacteriophage

• pour agar + E. coli mixture onto petri dishes containing bottom agar

• plates are incubated overnight

  • E. coli grows

  • phage infect E. coli cells and multiply

  • plaques formed (containing virions)

results of culture

• Cloudy area: growth of E.coli

• Cleared areas: plaques (E. coli killed by phage). spots where a virus has entered and ruptured

  • virions infect E. coli cells

  • plaques are formed upon the lysis of E. coli cells

  • one plaque is formed from infection of one virion: plaque forming units (pfu)