Lecture 12 - Bacteriophages, transduction, bacterial immunity Flashcards
What is a lytic phage? What is the structure and some examples?
- Circular chromosome copies self-assemble then cell lyses to release phages
- Head, tail, tail fibres, tail plate
- T2, T4, T7 of E coli
What is a lysogenic phage? What is the structure and some examples?
- Circular chromosome integrates in host genome as prophage to be passed on then enters lytic cycle after stress trigger
- Head + flexible tail
- Lambda, P1, P2, P4 of E coli, P22 of Salmonella
Who discovered transduction and how?
- Zinder and Lederberg
- S. typhimurium auxotrophs L22 with met and his, L2 with ar grown tog (shouldnt have)
- Not transformation bc DNase resistant, not conjugation bc no contact
- L22 had P22 phage
What is the 3 step process of DNA packaging into phages?
- Terminase recog end of phage DNA, binds portal protein into procapsid
- ATP to drive packaging
- Cleavage by recog sequence to get correct amount of DNA into capsid
What are concatemers?
2+ genome copies linked end-end formed during phage rep
Explain headful packaging using T4 phage as an example
- T4 = linear, 5 genes
- Terminally redundant and circular permutation = ABCDEA etc
- Bidirectional rep -> 3’ overhang hybridise to concatemer -> halved = 5’ overhang -> filled in and packaged
- 1 copy + 5kb each end
Explain site dependent packaging using P22/pac phage as an example
- P22 = circular
- Lambda/cos sites = 12nt repeats recog by cleavage enzyme
- Rolling circular rep + conjugation like F plasmid = nicked @ cos/lambda sites -> sstrand synthesis -> nicked again
Explain combination packaging. What are the 2 phages used as an example?
- Headful packaging but with specific pac sites
- 1 copy + 7-12% more
- P1 and P22 phages
What types of packaging can be used for generalised transduction and why?
- Headful = only have to recog 3’ end of chromosome
- Combo = pseudo cos/pac site motifs
What are the 2 requirements of generalised transduction? Include phage examples
- Phage can’t degrade host DNA = delete nucleases eg T4 good if no nuclease
- Degenerate pac sites in host recog by phage enzymes with lower specificity eg P1 and P22
What 3 reasons explain why generalised transduction is rare?
- Mistaken packaging rare
- Bacteial pac-like sites cut less efficiently by phage nucleases
- Transduced DNA doesn’t always survive
What are the 4 fates of DNA from generalised transduction?
- RecA homologous recombo into recipient + expression of new trait
- DNA reps in recipient
- DNA degraded by host restriction enzymes
- Abortive transduction = stable, can’t rep, 1 daughter inherited only, not understood
What is specialised transduction?
Induced prophage excised with adjacent host genome then repped and packaged so every phage has genetic marker
What are 2 examples and 2 reasons why transduction is significant?
- Eg1 = photosystems 1 and 2 in cyanobacteria from phages
- Eg2 = S pyogenes flesh eating bc nuclease from phage
1. DNA stable in phage = lasts longer
2. Transduction between distantly related possible
Why is foreign invading DNA bad for recipient cells?
Metabolic deficit to rep it
What 3 ways do bacteria protect themselves from foreign DNA?
- Mutations/alteration of phage binding sites on cell surface
- Restricion modification = restriction enzymes to cleave foreign, methyltransferases to protect DNA motifs
- CRISPR-Cas = RNA adaptive immune system to recog foreign DNA
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
What does Cas stand for and what does it do?
- CRISPR associated protein
- Helicase to unwind, nuclease to cut foreign DNA
What is the general structure of the CRISPR-Cas system?
Conserved hairpin loop repeats and variable protospacers from foreign DNA
What is the structure of CRISPR-Cas RNAs?
CAS genes, leader + promoter, CRISPR array
Explain the 3 step process of the CRISPR-Cas mechanism
- Leader + promoter activated = transcribe array to precursor RNA then CRISPR RNA with protospacer + protospacer associated motif (PAM
- CRISPR-RNA associates with Cas protein using PAM = effector complex to attach and scan foreign DNA for homology
- Unwind DNA @ homologous regions and cleave to block infection
How does the CRISPR-Cas system create memory of infection?
If cell survives foreign DNA, adds fragments of it to CRISPR array as memory to be passed on
