GENET 270 lec 5- phage
what is a bacteriophage
viruses that infect bacteria
How are all organisms on Earth parasitized by viruses?
All organisms on Earth are parasitized by viruses because viruses can infect every form of life — from bacteria (infected by bacteriophages) to plants, animals, fungi, and even other viruses. Viruses rely on the host’s cellular machinery to reproduce, meaning wherever there is life, there are viruses adapted to exploit it.
what is the most numerous bilogical entities on earth
phage particles--> infect 10^23 bac/sec
How does phage–bacteria evolution differ from human–bacteria evolution in timeline speed?
Phage–bacteria evolution happens much faster because both reproduce in minutes, leading to rapid coevolution. Human–bacteria evolution is slow since humans reproduce over decades.
How do phage antibacterial strategies differ from human antibacterial mechanisms?
Unlike antibiotics, phages are highly specific to bacterial strains, can evolve alongside bacteria, spare beneficial microbes, and self-replicate at infection sites.
Is the genome size of phages and viruses very diverse?
Yes. Viral genomes, including those of phages, are extremely diverse — they can range from just a few thousand bases (small RNA viruses) to over two million bases (giant DNA viruses). Phage genomes also vary widely depending on their host and lifestyle.
How do phage structures differ from other viruses?
Phages often have complex structures with an icosahedral head that holds DNA and a tail used to inject it into bacterial cells. Other viruses infecting plants or animals are usually simpler — often just a capsid (sometimes with an envelope) without tails.
How do icosahedral-headed, elaborate-tailed phages differ structurally from filamentous phages?
Icosahedral-tailed phages (like T4) have a distinct head and tail:
Head (capsid): Icosahedral shape enclosing double-stranded DNA.
Tail: A long, contractile or noncontractile tube with base plate and tail fibers used to attach and inject DNA into bacteria (break through membrane/cell wall). recognize specific recptor on host.
Filamentous phages (like M13) are long, flexible tubes:
Structure: Rod-shaped capsid made of repeating protein subunits wrapped around single-stranded DNA.
Key difference: They lack a separate head and tail — the DNA runs along the length of the filament (phage as long as genome), and they exit the host without lysing it.
Do icosahedral phages have a bigger host range than filamentous phages?
Generally, yes. Icosahedral phages—especially those with complex tails—tend to have a broader host range because their tail fibers and base plates allow specific receptor recognition across more bacterial strains.
Is there only a small number of phage families? are phages within a family closley related
Yes. There are only a few main phage families, but each contains many diverse species adapted to different bacterial hosts. yes
How do Myoviridae, Siphoviridae, and Podoviridae differ?
Myoviridae: Have long, contractile tails that inject DNA into the host by contracting like a syringe. (KS12)
Siphoviridae: Have long, flexible, noncontractile tails used to gently deliver DNA through the bacterial membrane. (KS9)
Podoviridae: Have short, stiff tails that attach closely to the bacterial surface for DNA injection. (DC1)
How do phage genomes show independent evolution from their bacterial hosts?
Phage genomes evolve independently because they frequently swap, lose, or gain genes through horizontal gene transfer and recombination, rather than coevolving strictly with the host’s genome. This creates mosaic, fast-changing genomes distinct from their bacterial hosts.
What is the mosaic nature of phage genomes, and how are phages assembled from shared “mosaic tiles”?
Phage genomes are mosaic, meaning they’re built from gene segments exchanged among different phages. These “mosaic tiles” are individual genes or functional modules (like DNA replication, capsid, or tail assembly genes) that can be swapped between phages through recombination. Over time, this mix-and-match process creates unique phage genomes assembled from shared genetic parts.
How do mosaic gene modules in phage genomes show synteny?
Synteny means genes appear in the same order across different phages. Even when gene sequences differ, the functional order of modules (like head, tail, lysis) is often conserved.
Do phage gene modules always occur in the same order?
Usually yes — core modules follow a common layout (e.g., replication → structural → lysis), though exact gene content varies.
Can gene order be conserved even when phages share little sequence similarity?
Yes. Even with low sequence homology, gene order stays similar because it reflects functional and temporal organization during infection.
Why does conserved gene order persist among diverse phages?
Because gene arrangement supports efficient assembly and timing of expression — early genes for replication, middle for structure, late for lysis — keeping infection orderly despite genetic diversity.
How does the mosaic nature of phage genomes arise?
It arises through recombination between phages, promoted by short homologous sequences flanking modules. These regions make it easy for DNA to swap during co-infection. Random recombination events create new gene combinations, and natural selection preserves the functional ones — leading to mosaic, mix-and-match genomes.
In random recombination, do protein products within a module interact?
Yes. Proteins within the same module often interact closely because they work together in a shared function (like building a tail or capsid).
In random recombination, can modules with proteins that can’t interact be combined?
Yes — random recombination can mix modules whose proteins don’t interact properly, but those phages are usually nonfunctional and eliminated by selection. Only combinations where protein interactions still work are retained and passed on.
how many phage families are there
13
What are the two pathways phages can follow?
Phages can follow either the lytic or lysogenic pathway.
What happens in the lytic pathway?
The phage quickly replicates inside the host, assembles new viruses, and lyses (bursts) the cell to release them — usually within 20–60 minutes.
What happens in the lysogenic pathway?
The phage integrates its DNA into the host genome and stays dormant as a prophage, replicating along with the bacterial cell.
Do these pathways have to be coordinated with cell division?
The lysogenic cycle is tied to cell division because the prophage replicates with the host DNA. The lytic cycle isn’t coordinated — it destroys the cell instead.
What is the main purpose of these pathways?
To ensure phage survival and propagation — the lytic cycle spreads phages rapidly (make the most copies of genome possible-100-1000 copies in 20/30min), while the lysogenic cycle preserves them long-term when conditions aren’t favorable for lysis.
How do icosahedral and tailed phages enter and exit bacterial cells?
Entry: They attach to specific receptors on the bacterial surface using tail fibers, then inject their DNA through the tail into the cell — the capsid stays outside.
Exit: After replication and assembly, phages use lysozyme-like enzymes to break the cell wall, causing lysis and release of new phages.
What are the six main steps of the phage lytic cycle?
Attachment (Adsorption): Phage binds to specific receptors on the bacterial surface.
Penetration: Phage injects its DNA into the host cell.
transcription: phage dna is trancribed producing phage mrna which is translated to phage proteins
replication: phage coat proteins, other protein components and dna are produced separately. host dna degraded.
assembly: phage assembled
Lysis (Release): The host cell wall is broken down, releasing new phages.
How do filamentous phages enter and exit bacterial cells? (F1, M13)
Entry: Filamentous phages attach to bacterial pili (like F pili) and slide their single-stranded DNA through the pilus into the cell (phage ingested by cell).
Exit: They don’t lyse the cell — new phage particles are secreted slowly through the membrane, allowing the host to survive and keep producing phages.
Why are icosahedral phages considered acute infections while filamentous phages are chronic?
Icosahedral phages cause acute infections because they quickly replicate, lyse, and kill the host cell.
Filamentous phages cause chronic infections since they continuously release phage particles without killing the host, leading to long-term, nonlethal infection.
What are the six steps of the chronic infection cycle of filamentous phages?
Attachment: The phage attaches to bacterial pili (like the F pilus) on the cell surface.
Entry: The phage’s single-stranded DNA (ssDNA) genome enters the host through the pilus channel.
Conversion: Inside the host, host DNA polymerase converts the ssDNA into a double-stranded replicative form (RF DNA).
Replication: The phage protein g2p nicks one strand of the RF DNA to start rolling-circle replication. Host DNA polymerase extends from the nick, synthesizing new ssDNA genomes while displacing old ones.
Coating & Protection: The displaced ssDNA is bound by g5p proteins, which protect it and prevent premature packaging.
Assembly & Secretion: The ssDNA–g5p complex is directed to the cell membrane, where it’s coated with phage coat proteins and secreted through the membrane. The host cell isn’t lysed and continues releasing phages chronically.
How do DNA polymerase III and exonuclease activity create RF (replicative form) DNA in filamentous phages?
After the phage’s single-stranded DNA (ssDNA) enters the host, host DNA polymerase III synthesizes the complementary strand, using RNA primers to start replication. Then, exonuclease enzymes remove the RNA primers, and DNA ligase seals the gaps. The result is double-stranded circular DNA (RF DNA) — the template used for further replication and transcription.
What does Gene II endonuclease do when it nicks the + strand origin and forms a covalent linkage to the 5′ end?
Gene II (g2p) makes a nick at the origin of replication on the + strand of RF DNA and becomes covalently attached to the 5′ end. This creates a free 3′-OH group for DNA polymerase to extend, starting rolling-circle replication to make new single-stranded phage genomes.
what happens after the nick
Host DNA polymerase adds new nucleotides, using the intact strand as a template and pushing the old strand off as a new single-stranded DNA.
steps of rolling circle replication
Nick & Attachment: g2p (Gene II protein) cuts one strand of the circular RF DNA (the + strand) at the origin of replication and stays attached to the 5′ end covalently via Tyr of the nicked strand.
Template Use: host rep helicase unwinds dna at nick, The intact (–) strand acts as a template. DNA polymerase starts adding new nucleotides from the 3′-OH created by the nick.
Displacement: As polymerase moves around the circle, it pushes the old + strand off — this is the new single-stranded DNA (ssDNA) genome being synthesized. which is resealed by gpII (transesterification)
Reconnection: When the polymerase completes a full circle, g2p cleaves and re-ligates the DNA — sealing the original RF molecule back into a circle.
Repeat: g2p can then nick again, starting another round of rolling-circle replication to make more ssDNA copies.
stop: continues until gene v product accumulates--> coats = strand, prevents RF synthesis
Why does rolling-circle replication continue until Gene V product accumulates?
Because Gene V product (g5p) acts as a regulator that signals the cell to switch from making double-stranded RF DNA to making single-stranded genomes for packaging.
What is the Gene V product?
g5p is a single-stranded DNA-binding protein that coats and stabilizes the new ssDNA genomes, forming a g5p–ssDNA complex.
How does g5p prevent further RF synthesis?
When g5p binds ssDNA, it blocks it from being converted back into RF DNA by DNA polymerase. This shifts the balance from replication of RF templates to production and packaging of ssDNA phage genomes.
What is the main problem when replicating linear phage genomes?
The main problem is the end-replication issue — DNA polymerases can’t fully copy the very ends of linear DNA strands. This leaves unreplicated gaps or loss of terminal sequences each cycle, which can shorten or damage the genome unless the phage uses special strategies (like terminal proteins, hairpin loops, or circularization) to protect and complete the ends.
Why can’t DNA replication reach the ends of linear phage genomes?
Because DNA polymerase needs a 3′-OH primer to add new nucleotides. On the lagging strand, the final RNA primer near the end is removed, but there’s no upstream 3′-OH for DNA polymerase to fill in the gap. As a result, the very ends stay unreplicated, causing the end-replication problem.
How do eukaryotes solve the end-replication problem?
They use telomerase, which adds repetitive DNA (telomeres) to chromosome ends using an RNA template, preventing gene loss.
How do phages solve the end-replication problem?
Phages use several strategies:
ircularization: The linear DNA joins its ends to form a circle, removing the problem entirely.
Protein primers: Specialized terminal proteins provide a 3′-OH group for DNA polymerase to start synthesis at the ends.
Terminal redundancies / concatemers: Repeated end sequences allow replication overlap and proper genome packaging.
Hairpin ends: The DNA folds back on itself to form double-stranded loops, giving polymerase a built-in primer to copy the ends.
What is terminally redundant DNA?
It means the ends of a phage’s DNA molecule contain repeated sequences — the same short stretch of DNA appears at both ends.
Why do phages have terminal redundancy?
When the DNA replicates and gets packaged into new phages, these repeated ends can overlap and join with other copies, forming long continuous DNA chains (concatemers).
This overlap makes sure no genetic information is lost at the ends during replication or packaging.
How are individual genomes cut out of a concatemer?
Specialized phage enzymes called terminases (endonuclease) cut the concatemer into individual genome-length pieces starting at pac sites .
The small terminase subunit recognizes specific DNA packaging sites.
The large terminase subunit cuts the DNA and powers its insertion into the phage head (capsid).
How does the T7 phage use this mechanism, and does it use its own machinery?
Yes — T7 phage also forms concatemers and uses its own phage-encoded enzymes (nucleases to degrade host dna) to process them.
T7 DNA replication produces concatemers via recombination and incomplete termination of replication.
The T7 terminase complex then cuts and packages one genome-length piece per capsid, ensuring each phage receives a complete genome.
How does T4 concatemer synthesis differ from T7?
T4 phage: Uses recombination-dependent replication — multiple replication forks merge and recombine, forming long concatemers with terminal redundancy. Its replication is continuous and driven by recombination and repair-like processes. 30 T4 gene products requried, unreplicated terminally redudnat 3' ends invade daughter dnas to create recomb intermediates that prime replication.
T7 phage: Uses unidirectional replication from a fixed origin, producing concatemers mainly through incomplete termination and rejoining of ends. It’s more controlled and sequence-specific, guided by its own T7 DNA polymerase and terminase.
What does “hijacked RNAP” mean in T4 replication?
It means T4 uses the host’s RNA polymerase to make short RNA primers for its own DNA replication.
How is T4 primer synthesis unique?
Instead of a primase, T4 uses host RNAP, modified by phage proteins, to transcribe short RNAs near origins that act as primers for T4 DNA polymerase.
How does T4 modify host RNA polymerase (RNAP)?
T4 uses AsiA and MotA (gp55) proteins to modify host RNAP.
What does AsiA do?
AsiA binds the host sigma factor (σ⁷⁰) and blocks it from recognizing normal E. coli promoters, disabling host transcription-> only allowing t4 transcription
What does MotA (gp55) do?
MotA (gp55) replaces the host sigma function and redirects RNAP to T4 middle promoters, ensuring phage genes are transcribed.
How does this lead to replication initiation?
The modified RNAP transcribes short phage RNAs that invade dsDNA and base-pair with complementary sequences, forming R-loops. These R-loops act as primers for T4 DNA polymerase to start replication.
Why does T4 replication initiate at multiple origins (oris)?
Because T4 needs to replicate its large genome very quickly. Starting replication at multiple origins allows many replication forks to form at once, speeding up DNA synthesis and ensuring the entire genome is copied before the host cell is lysed.
how does t4 dna replication differe from chromosomal rep
only through the replciation at multiple oris
What happens after initiation at multiple origins in T4 replication?
The next step is formation of recombination-dependent replication (RDR), which takes over once origin-based replication stalls.
How are D-loops created?
When 3′ single-stranded DNA ends (made because DNA polymerase can’t fully copy linear ends) invade homologous regions of another DNA molecule, they form D-loops — short regions where one strand of DNA displaces another and pairs with its complement.
How do D-loops differ from R-loops?
R-loops: Formed by RNA invading double-stranded DNA.
D-loops: Formed by DNA invading double-stranded DNA.
R-loops: Formed by RNA invading double-stranded DNA.
D-loops: Formed by DNA invading double-stranded DNA.
They’re produced because DNA polymerase can’t replicate the linear DNA ends, and by viral gp46 and gp47 nucleases, which function like the bacterial RecBCD complex to resect DNA ends for recombination.
How does the D-loop work in T4 replication?
The viral UvsX protein (analogous to RecA) promotes pairing between the 3′ single-stranded DNA end and a homologous region in another DNA molecule, displacing one strand to form a D-loop (DNA invasion loop).
What roles do UvsX and UvsY play?
UvsY helps UvsX by removing gp32, the T4 single-stranded DNA–binding protein, from the ssDNA so UvsX can bind and initiate strand invasion.
What happens after the D-loop forms?
The T4 DNA helicase is loaded onto the invaded strand, and leading-strand synthesis begins from the 3′ end. The T4 primase then makes primers on the displaced DNA strand, starting lagging-strand synthesis — creating a new replication fork.
What is unusual about this D-loop–based replication process in T4?
It’s unusual because replication starts through recombination, not at a fixed origin. Instead of needing origin primers, a 3′ DNA end invades another molecule to form a D-loop that acts as the primer for DNA synthesis. This recombination-dependent replication (RDR) allows T4 to keep replicating even after origin-based replication stops or DNA damage occurs — a unique and flexible backup system.
What is meant by headful packaging in bacteriophage T4?
It’s a DNA packaging mechanism where the phage fills its capsid (“head”) with slightly more DNA than one complete genome length. Once the head is full, the DNA is cut and packaging stops.
Why does T4 DNA packaged by the headful mechanism have terminally redundant ends?
Because each head is filled with more DNA than one genome’s worth, so extra sequences are added to the ends — these sequences are repeats (terminal redundancies) of parts of the genome.
What are cyclically permuted genomes in T4 DNA?
Each packaged genome starts and ends at different points in the original concatemer but contains the same overall genetic information. The gene order is “rotated” compared to the standard reference genome.
What are cyclically permuted genomes in T4 DNA?
Each packaged genome starts and ends at different points in the original concatemer but contains the same overall genetic information. The gene order is “rotated” compared to the standard reference genome.
What is a concatemer, and what role does it play in T4 headful packaging?
A concatemer is a long continuous DNA molecule containing multiple genome copies linked end-to-end. The packaging machinery cuts and loads these sequentially into capsids until each one is full.
Where do the vertical arrows on T4 DNA packaging diagrams typically point?
They indicate the sites of cleavage — where the packaging machinery cuts the concatemer once a head is full of DNA.
How does headful packaging contribute to genetic diversity among T4 phage particles?
Because each phage head receives a genome with a different cyclic permutation, the linear DNA ends differ slightly, producing genetic variation in genome arrangement (though not in content).
How does diversity in genome arrangement differ from diversity in genome content, and why is this useful in T4 phage?
Arrangement diversity means the same genes appear in a different linear order (cyclic permutations), while content diversity means actual differences in genetic information. In T4, arrangement diversity helps efficient packaging and recombination, without changing the genetic content or phenotype.
What is phage lysis?
It’s the process by which an acute infection phage breaks open (lyses) the bacterial host cell to release newly produced progeny phages.
What type of infection is associated with phage lysis?
Acute infections — phages rapidly infect, replicate, and then lyse the host cell to release progeny.
What are the two known systems of phage lysis?
Single-protein lysis system
Timed (holin-dependent) lysis system
What are holins, and why are they important in phage lysis?
Holins are phage-encoded proteins that form pores in the bacterial cell membrane at a specific time during infection, allowing other lysis enzymes (like endolysins) to access and degrade the cell wall.
🧠 Think of holins as molecular “timers” that decide when the cell bursts.
How does single-protein lysis work?
The phage produces a single protein that binds to enzymes responsible for synthesizing peptidoglycan (PG) precursors. When these precursors run out, no more cell wall can be made, leading to cell rupture and phage release.
What is an example of a phage that uses single-protein lysis?
φX174 (Phi X174) — a very small bacteriophage that encodes a single lysis protein.
Why is single-protein lysis inefficient compared to holin-dependent lysis?
Because the number of progeny phages produced depends on when in the bacterial cell cycle the infection occurs — if the cell is near the end of wall synthesis, fewer phages are made before lysis.
Why might some small phages evolve to use single-protein lysis instead of holins?
Because small phages have limited genome size and can’t encode multiple lysis proteins; a single multifunctional protein saves genetic space, even if it’s less efficient.
What is the purpose of timed (holin-dependent) lysis in phages?
To allow the phage to produce the maximum number of viral particles before bursting the host cell at an optimal time.
How many proteins are typically required for timed lysis?
At least five proteins — including holin, anti-holin, endolysin, and two spanins (Rz and Rz1).
What is the function of endolysin (lysozyme) in timed lysis?
Endolysin degrades the bacterial cell wall’s peptidoglycan (PG), enabling cell rupture once it reaches the wall.
What is the role of the two spanins (Rz and Rz1)?
They assist in disrupting or separating the outer membrane from the cell wall during the final stage of lysis, completing the cell’s breakdown.
🧠 Spanins = "spanners" between wall and outer membrane.
What is the function of holins in timed lysis?
Holins form pores in the inner (cytoplasmic) membrane, allowing endolysin to pass through and reach the peptidoglycan layer.
They can also collapse the membrane potential, triggering cell lysis.
What are anti-holins, and what do they do?
Anti-holins inhibit holin activity until the correct time for lysis, preventing premature cell death and allowing complete viral assembly.
In the λ (lambda) phage, which gene encodes the holin–anti-holin system?
The S gene encodes both proteins:
S105 = Holin
S107 = Anti-holin
How do the holin and anti-holin differ in structure?
Anti-holin (S107) has two extra amino acids at the N-terminus compared to holin (S105). These prevent the N-terminal α-helix from inserting into the membrane, keeping the holin inactive.
How does anti-holin inhibit holin activity?
Anti-holin dimerizes with holin to block pore formation, keeping endolysin trapped inside until lysis timing is optimal.
What triggers holin activation and pore formation?
When the membrane depolarizes (by an unknown mechanism), the N-terminal α-helix of anti-holin inserts into the membrane, allowing holin pores to form and endolysin to be released.
Why is the holin–anti-holin system more efficient than single-protein lysis?
It precisely controls lysis timing, ensuring that the host cell bursts only after the maximum number of phage particles has been assembled.
Explain how the S105 and S107 proteins coordinate lysis timing in lambda phage.
Both are made from the same gene (S).
S107 (anti-holin) has two extra N-terminal amino acids preventing its insertion into the membrane.
It binds to S105 (holin), blocking pore formation.
When the membrane depolarizes, anti-holin changes conformation, enabling S105 to form pores → endolysin released → cell lysis.
What normally triggers lysis in the holin–anti-holin system?
A change (collapse) in membrane potential — this allows holins to insert into the membrane, form pores, and release lysozyme (endolysin) to degrade the peptidoglycan.
What happens in a normal lysis event when no anti-holin is present?
Holins freely insert into the inner membrane, form pores, and allow lysozyme to reach and degrade the peptidoglycan → cell lysis and phage release.
What is lysis inhibition (LIN) in T4 phage infections?
It’s a delay in lysis caused when a second T4 phage infects a cell that’s already infected. The newly injected T4 DNA stabilizes the anti-holin, preventing pore formation and lysis.
What happens to the DNA of the second T4 phage during lysis inhibition?
The incoming T4 DNA does not enter the cytoplasm — it remains in the periplasm, where it helps stabilize the anti-holin protein.
How does T4 anti-holin function during lysis inhibition?
It binds to and inactivates holin, blocking pore formation in the membrane and preventing the release of lysozyme/endolysin.
What is the biological purpose of lysis inhibition in T4 phages?
It prevents the host cell from lysing when many phages are already nearby — allowing time for more phage replication inside before release.
🧬 It’s a “wait until the coast is clear” strategy.
What ecological or experimental observation suggests lysis inhibition is occurring?
Phage plaques do not fully clear on a bacterial lawn — because infected cells are delaying lysis, reducing visible clearing.
lysis inhibition mechanism (6 steps)
Step Event
1 First T4 infects → produces holin, anti-holin, and endolysin
2 Normally, when membrane potential collapses → holin inserts → lysis
3 Second T4 infects same cell
4 Second phage DNA stays in periplasm and stabilizes anti-holin
5 Anti-holin binds holin → no pore formation
6 Endolysin trapped → lysis delayed (lysis inhibition)
Why is lysis inhibition considered an evolutionary advantage for T4 phages?
It allows phages to maximize replication within an already infected cell instead of destroying it prematurely when other phages are nearby — conserving host resources and preventing competition.