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1: GENET 270 > Phages > Flashcards

Phages Flashcards

1
Q

what are bacteriophages?

A

-viruses that infect bacteria, ancient, most numerous biological entities on Earth

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2
Q

Phage - Bacteria evolution far preceded ______ evolution.

A
  • Human - Bacteria
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3
Q

what is the genome size of bacteriophages?

A

2,435 bp to 316,674 bp; usually ~40,000 bp (bigger phages more complex)

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4
Q

What are the two structures of bacteriophages?

A
  • icosahedral heads
    -filamentous
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5
Q

what is the structure of icosahedral head bacteriophages?

A
  • has capsids to encapsulate tightly packed genome
  • tails recognize specific receptor on host -> host range
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6
Q

what is the structure of filamentous bacteriophages?

A

long filaments that lack heads/tails and the phage is as long as genome

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7
Q

Phage families are characterized by 3 things

A

morphology and DNA/RNA, & linear vs circular nucleic acid.

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8
Q

what are caudovirales?

A

class of phages that are tailed

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9
Q

What are three types of caudovirales? what are their characteristics?

A

Myoviridae (linear dsDNA, large phage, contractile tail)
Siphoviridae (linear dsDNA, long tail, tail fibers)
Podoviridae (linear dsDNA, short tail)

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10
Q

how do we know that phages underwent independent evolution?how do phages evolve then?

A
  • sequence bear little homology to bacterial host
    -by exchanging modules with family members
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11
Q

Phages have a mosaic nature meaning that theu are assembled from shared mosaic tiles (show synteny). What are mosaic tiles?

A

-genetically linked genes (individual gens or modules of genes for same function); retain the same order even when in different phages

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12
Q

How does the mosaic nature of phages come about?

A

1) Sequences flanking modules promote recombination
2) Random recombination and selection (modules must still work or it will be selected against)

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13
Q

why is module synteny useful for a phage?

A

-genes need to be together in same transcript or gene cluster
-genes need to be expressed at same time during phage life cycle

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14
Q

phage genome replication & packaging does not have to be coordinated with this cell process?

A

cell division

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15
Q

phage can make how many copies of genome in 20-30 minutes?

A

100s-1000s

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16
Q

Icosahedral & tailed phages cause which type of infection?

A

lytic (acute) infection

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17
Q

How do Icosahedral & tailed phages enter and exit cell?

A

1) Attachment to receptors on cell wall
2) Penetration: phage DNA injected & protein coat left outside
3) Transcription: phage DNA is transcribed -> phage mRNA -> translated to phage proteins
4) Replication of phage DNA & Synthesis -> protein coat proteins, other proteins, phage DNA replicated; host DNA degraded
5) Assembly: phage components are assembled into mature virions
6) Release: bacterial cell lyses & release infective phages

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18
Q

Filamentous phages cause which type of infection?

A

chronic

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19
Q

How do filamentous phages enter and exit cell?

A

-adsorb to bacterial pilus
- phage ingested by cell
- protein coat removed
- after replication & packaging, phage leaks out through membrane; no lysis of cell

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20
Q

What are three examples of circular ssDNA phages?

A

X174- spherical capsid with spikes
M13 & F1 - filamentous

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21
Q

how are circular ssDNA phages replicated? (3 steps)

A

1) First a dsDNA replicative form is synthesized (rf- replicative form)
2) Then rolling circle replication used to generated many ssDNA genomes but instead of Rep protein nicking, it is Gene II product protein
3) This continues until gene V product accumulates and coats + strand to prevent RF synthesis

22
Q

what kind of genome do most phages have?

A

linear genome that circularizes in host

23
Q

what is the problem with linear genomes?

A

when rep fork reaches end, no way to synthesize primer upstream to complete synthesis of lagging strand

24
Q

How do eukaryotes solve the linear genome problem?

A

telomerases synthesize “throw away DNA” at telomeres at end of replication

25
Q

What are the 4 ways phages solve the linear genome problem?

A

1) circularize after infection (has complementary ends that can H-bond)
2) protein primers
3) terminal redundancies & concatemers
4) hairpin ends

26
Q

what is terminally redundant DNA?

A

both ends are the same

27
Q

How are T7 linear phage genome replicated? (4)

A

1) T7 uses its own polymerase, ligase, helicase, rpimase and RNAP and encode nucleases to degrade host DNA
2) Genome contains terminal redundancies at ends so after replication, the unreplicated 3’ ends pair
3) This generates concatemers which are individual genomes linked end-to-end
4) individual genomes arecut out of concatemer by
endonuclease at pac sites

28
Q

How are T4 linear phage genome replicated?

A
  • Similar to T7, it also synthesizes concatemers, but
    uses recombination and requires 30 T4 gene products
29
Q

2 steps of T4 phage genome replication

A
  1. replication initiates from ori’s throughout the genome
  2. unreplicated, terminally redundant 3’ ends invade daughter DNAs and create recombination intermediates that prime replication to form large concatemers (RDR – recombination directed replication)
30
Q

How does T4 phage initial primer synthesis happen during DNA replication?

A

1) host RNAP are modified with MotA (gp55) & AsiA proteins and recognize viral middle promoters
2) transcription is initiated
3) mRNAs invade dsDNA and pair to complementary sequences, creating “R loop” (ssRNA invasion of dsDNA) to prime replication

31
Q

How does AsiA modify RNAP? What about MotA?

A

-AsiA blocks E.coli Sigma from binding E.coli
-MotA replcaces E.coli Sigma to direct RNAP to phage promoters

32
Q

Generally, what is recombination-dependent replication?

A

leading strand replication is primed by recombination intermediates as DNA is invaded by ssDNA 3’OH (D loop)

33
Q

For RDR: 3’ ssDNA ends are generated by two things?

A

1) inability of DNA pol to replicate ends of linear DNA
2) viral gp46 & gp47 nucleases (analogous to RecBCD)

34
Q

How does strand invasion by ssDNA 3’OH (Dloop) allow for RDR to happen (T4 phage)?

A

1) D loop pairing is promoted by viral UvsX = analogous to RecA
2) UvsX & UvsY act to displace gp32 (T4 ssDNA binding protein)
3) T4 DNA helicase loaded
4) leading strand synthesis initiated from invading 3’ OH DNA
5) T4 primase initiates lagging strand synthesis on displaced DNA

35
Q

Packing of DNA longer than a single genome equivalent gives rise to ________ & _______; this happens with T4

A

-repeated terminally redundant ends
-cyclically permuted genomes

36
Q

headful packaging happens how? what is the result on the ends on the genome?

A

cutting from concatamers; varying ends

37
Q

acute infection phages do what? require what type of proteins?

A

lyse host cell to release progeny (require holins)

38
Q

What are the two systems for phage lysis for acute infection phages

A

1) single protein lysis (for very small phages - X174)
2) timed lysis

39
Q

How does single protein lysis works?

A

1) a single protein bind to enzymes involved synthesizing peptidoglycan precursors
2) when the precursors run out, no more cell wall made and cell lyses

40
Q

what is the disadvantages of single protein lysis?

A

inefficient because # phage produced dependent on
when in bacterial cell cycle infection occurs

41
Q

what is the benefit of timed lysis?

A

allows production of maximum viral particles

42
Q

5 proteins involved in timed lysis. What are they?

A

1) endolysin/lysozyme: lyses bacterial cell wall (PG)
2) spanins (Rz, Rz1): lipoprotein & protein that spans periplasm, may assist in separating outer membrane from cell wall
3)holin: form holes in membrane to allow lysozyme release OR that destroy membrane potential & cause cell lysis, multiple membrane domains
4) anti-holin: prevents activity of holin until appropriate time

43
Q

lysozyme/endolysin work in the ______. holin/antiholin works in _____

A

-periplasm/cytoplasm
-inner membrane

44
Q

Endolysins (or lysozyme) are made ______ but kept inactive by the action of ______, preventing endolysins from reaching the cell wall.

A

-early in infection
- anti-holins

45
Q

Lambda phage encodes holin from s gene. What protein is made? An anti-holin is made from same gene but with what change?

A

-S105 protein
-extra 2AA at N-terminus (S107 protein)

46
Q

Lambda Phage holin vs anti-holin

A
  • The holin made from s gene -> S105 protein
  • anti-holin made from same gene, extra 2 AA at N-terminus -> S107
47
Q

For the lambda phage, anti-holin/holin pore is formed. Why? What happens instead?

A

-because N-terminal a-helix of anti-holin does not enter membrane
-instead anti-holin dimerizes with holin to prevent its activity

48
Q

For Lambda Phage, how does pore form in membrane?

A

when the membrane depolarizes, the N-terminal a-helix of anti-holin inserts into membrane & pore is made by holin and lysozyme can now enter periplasm

49
Q

For T4, how does timed lysis happen?

A

-unstable antiholin interacts holin inhibiting pore formation
-over time, anti-holin degrades
-when no antiholin, holins insert in membrane, form pore and lysozyme goes to PG

50
Q

What happens if another T4 phage infects a cell that is already infected with T4?

A

the newly injected T4 DNA does not reach the cytoplasm but it stays in periplasm and stabilizes the unstable anti-hollin

51
Q

What does stabilized anti-holin do to the T4 holin?

A

interacts with T4 holin and prevents holin from forming a pore to release/endolysin, causing a delay in progeny phage (aka lysis inhibition)

52
Q

what is the benefit of lysis inhibition?

A

prevent release of additional T4 phages when there are plenty around (ex. edge of plate)

1: GENET 270 (15 decks)

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