12 - Transduction and immunity Flashcards
Types of bacteriophage
Virulent and temperate phages
Virulent phages
Lyse and kill host cells (e.g. E.coli T2)
Temperate phages
Genetic material can remain within the host cell for a period without killing it (e.g. Phage lambda of E. coli)
Bacteriophage lifecycles
Lytic cycle (cell lyses releases phages) or lysogenic cycle (phage DNA integrates into chromosome, doesnt kill)
Discovery of transduction
- 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)
Transduction steps
- 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
Abortive transduction
Phage DNA is present but not expressed
Characteristics of generalised transduction
- Transducing phage contains only bacterial DNA
- Any bacterial host gene can be transduced
- Size of phage head determines amount of transduced DNA
Mechanism of transduction
- 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
Packaging of nucleic acid in the phage head
- 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
Concatemers
Two or more copies of phage genome linked end to end
3 ways that phages package DNA
- Headful mechanism (phage T4)
- Site dependent packaging (P22 phage)
- Combination packaging mechanism (P1 phage)
Circular permutation in headful mechanism
Same number of genes organised differently
Terminal redundancy in headful mechanism
Repeats at each end but not the same repeats between different genomes (AA, BB etc)
Concatemer synthesis from linear phage genomes
- 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
Site dependent packaging
- 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
Combination packaging mechanism
Nuclease will make a initial cut at pac site followed by headful packaging of 5-10 phages, then another cut followed by packaging
Example of combination packaging
- 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”
Headful packaging in transduction
Phage enzymes recognise free 3’ end of bacterial chromosome and package it
Two criteria required for phage to be capable of generalised transduction
- Phage must not degrade host DNA completely after infection
- Degenerate pac sites on bacterial chromosome must be recognised by phage enzymes
Combination packing in transduction
Pac/cos-like sites occur in bacterial chromosomes too, although less frequently
Why is generalised transduction rare
- Due to mistaken packaging of bacterial DNA
- Bacteria posses fewer pac sites
- Transduced DNA must survive in recipient cell
Cotransducible
Genes close enough together to be carried within the same phage particle
Possible fates of transduced bacterial DNA
- Recombination into recipient genome (homology dependent)
- DNA replicates in recipient
- DNA degraded
- Abortive transduction (occurs 90% of time)
Requirements of specialised transduction
- 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
Results of specialised transduction in recipient
- 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
Bacterial immunity to phages and plasmids
- Mutation or alteration of the bacteriophage binding target site on the bacterial surface
- Two mechanisms (restriction modification enzymes and CRISPR-Cas)
Restriction modification enzymes
Restriction enzyme which cleaves incoming foreign DNA in concert with a methyltransferase which protects native DNA
CRISPR-Cas systems
RNA-directed adaptive immune systems in many
bacteria that recognize nucleic acids of invading plasmids and viruses.
What does CRISPR stand for
Clustered regularly interspaced short palindromic repeats
What does cas stand for
CRISPR associated protein
CRISPRs
Short repetitions of base sequences which are separated by short ‘spacer DNA’ from previous exposure
Cas
Has helicase activity to unwind DNA and nuclease activity to cut DNA
Molecular events of CRISPR-cas system
- 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.
