12 - Transduction and immunity

Question 1

Types of bacteriophage

Answer 1

Virulent and temperate phages

Question 2

Virulent phages

Answer 2

Lyse and kill host cells (e.g. E.coli T2)

Question 3

Temperate phages

Answer 3

Genetic material can remain within the host cell for a period without killing it (e.g. Phage lambda of E. coli)

Question 4

Bacteriophage lifecycles

Answer 4

Lytic cycle (cell lyses releases phages) or lysogenic cycle (phage DNA integrates into chromosome, doesnt kill)

Question 5

Discovery of transduction

Answer 5

• Two strains were mixed and produced prototrophs
• Occured in presence of DNase (not transformation)
• Occured when strains were separated by filter (not conjugation)
• Phage was present (transduction)

Question 6

Transduction steps

Answer 6

• Destruction of host DNA
• Synthesis of virus DNA and coat proteins
• Virus capsid synthesis and virus assembly
• Lysis of cell with release of phage particles and subsequent infection of another cell

Question 7

Abortive transduction

Answer 7

Phage DNA is present but not expressed

Question 8

Characteristics of generalised transduction

Answer 8

• Transducing phage contains only bacterial DNA
• Any bacterial host gene can be transduced
• Size of phage head determines amount of transduced DNA

Question 9

Mechanism of transduction

Answer 9

• Both virulent and temperate phages can mediate generalised transduction, as both have lytic cycle of replication
• Bacterial DNA packaged into phage head instead of viral genome

Question 10

Packaging of nucleic acid in the phage head

Answer 10

• Terminase protein recognises the end of the phage DNA
• Terminase binds to the portal protein in the empty pro capsid
• ATP is consumed to drive nucleic acid packaging process in capsid
• A recognition sequence in the phage DNA is cleaved so that only the correct amount of DNA is inside the capsid

Question 11

Concatemers

Answer 11

Two or more copies of phage genome linked end to end

Question 12

3 ways that phages package DNA

Answer 12

1. Headful mechanism (phage T4)
2. Site dependent packaging (P22 phage)
3. Combination packaging mechanism (P1 phage)

Question 13

Circular permutation in headful mechanism

Answer 13

Same number of genes organised differently

Question 14

Terminal redundancy in headful mechanism

Answer 14

Repeats at each end but not the same repeats between different genomes (AA, BB etc)

Question 15

Concatemer synthesis from linear phage genomes

Answer 15

• T4 has linear DNA, that never circularizes.
• Replication of its linear DNA begins at specific origins and proceeds bi-directionally
• The 3’ ends can invade other molecules of DNA
  where there is homology, to produce linear concatemers
• Concatemers are cut in two to produce 5’ overhangs that are filled in and used in further rounds of replication
• The concatemers are packaged by length

Question 16

Site dependent packaging

Answer 16

• Lambda phage genome has 12 nt repeat (cos sites) on each end.
• Complementary ends bind to produce a circular genome.
• Each circular genome produces concatemers by Rolling circle replication
• cos sites are recognized by phage enzyme, cut, DNA packaged
• Highly specific

Question 17

Combination packaging mechanism

Answer 17

Nuclease will make a initial cut at pac site followed by headful packaging of 5-10 phages, then another cut followed by packaging

Question 18

Example of combination packaging

Answer 18

• P1 has linear dsDNA.
• After infection, the linear DNA forms a circle, that replicates and form concatemers.
• The DNA is packed into the phage heads by the “headful mechanism”

Question 19

Headful packaging in transduction

Answer 19

Phage enzymes recognise free 3’ end of bacterial chromosome and package it

Question 20

Two criteria required for phage to be capable of generalised transduction

Answer 20

• Phage must not degrade host DNA completely after infection
• Degenerate pac sites on bacterial chromosome must be recognised by phage enzymes

Question 21

Combination packing in transduction

Answer 21

Pac/cos-like sites occur in bacterial chromosomes too, although less frequently

Question 22

Why is generalised transduction rare

Answer 22

• Due to mistaken packaging of bacterial DNA
• Bacteria posses fewer pac sites
• Transduced DNA must survive in recipient cell

Question 23

Cotransducible

Answer 23

Genes close enough together to be carried within the same phage particle

Question 24

Possible fates of transduced bacterial DNA

Answer 24

1. Recombination into recipient genome (homology dependent)
2. DNA replicates in recipient
3. DNA degraded
4. Abortive transduction (occurs 90% of time)

Question 25

Requirements of specialised transduction

Answer 25

• Prophage formation
• When prophage is induced, errors are made in cutting the prophage out of the host
• Carries adjacent host genome with the phage genome
• Creates transducing particles

Question 26

Results of specialised transduction in recipient

Answer 26

• Crossover to integrate the bacterial genes in the genome, leaving intact copy of the phage genome
• Creation of a prophage containing both viral and donor DNA

Question 27

Bacterial immunity to phages and plasmids

Answer 27

• Mutation or alteration of the bacteriophage binding target site on the bacterial surface
• Two mechanisms (restriction modification enzymes and CRISPR-Cas)

Question 28

Restriction modification enzymes

Answer 28

Restriction enzyme which cleaves incoming foreign DNA in concert with a methyltransferase which protects native DNA

Question 29

CRISPR-Cas systems

Answer 29

RNA-directed adaptive immune systems in many
bacteria that recognize nucleic acids of invading plasmids and viruses.

Question 30

What does CRISPR stand for

Answer 30

Clustered regularly interspaced short palindromic repeats

Question 31

What does cas stand for

Answer 31

CRISPR associated protein

Question 32

CRISPRs

Answer 32

Short repetitions of base sequences which are separated by short ‘spacer DNA’ from previous exposure

Question 33

Cas

Answer 33

Has helicase activity to unwind DNA and nuclease activity to cut DNA

Question 34

Molecular events of CRISPR-cas system

Answer 34

• Full-length pre-crRNA is transcribed and processed into specific small RNA molecules that correspond to a
  spacer flanked by two partial repeats (cr-RNA).
• Each cr-RNA contains a protospacer which binds a matching sequence in foreign nucleic acid and a protospacer associated sequence (PAM) which binds Cas
• crRNAs bind to Cas proteins to form the effector complex.
• When the effector complex binds the incoming foreign nucleic acid, the complex activates to cut and degrade the foreign DNA or RNA.