Lecture 11 - Transformation, plasmids, conjugation Flashcards
Define core, accessory and pan genomes. What does closed and open pan genome mean?
Core = in all strains
Accessory = many not all
Pan = sum of all strains, closed if no HGT, open if HGT
Define HGT
Transfer of genes from 1 mature independent to another
What sort of DNA is transferred by transformation, conjugation and transduction?
- Transformation = free/naked DNA
- Conjugation = DNA by contact
- Transduction = DNA in phage
What does 2 things does transformation require?
- Linear/circular free/naked dsDNA
- Competent receiver = bind, import, protect from nucleases, competence proteins from regulated phenotype
What are the 2 fates of transformation?
- Degraded to components
- Integrated by non-reciprocal recombo into chromosome
List the 5 transformation machinery components in G-ve vs G+ve
- OM channel = G-ve PilQ
- CW channel = G-ve PilE, G+ve ComGC
- Irreversible DNA binding = G-ve ComE, G+ve, ComEA
- Nuclease N
- PM channel = G-ve ComA, G+ve ComFA
Explain the 5 step process of G+ve transformation
- Random collision = thru ComGC, ComEA bind DNA reversibly, DNase sensitive
- DNA fragmentation by endonuclease NucA
- Eclipse = ds->ss by N, irreversibly bound, DNase resistant
- Into cell by ComEC + ComFA translocases
- Non-reciprocal recombo by RecA coating ssDNA, pairing with host to triple strand, endonuclease nicks both, normal repair = chance
When is transformation more efficient and why?
Closely related bc more homology
Describe the features and types of plasmids
- Circular supercoiled dsDNA replicons in cytoplasm independent of chromosomal replication, 2-500kB, accessory genome
- Fertility, resistance, virulence, metabolic types
How was the F plasmid discovered?
- Lederberg and Tatum
- E coli prototrophs and mutant auxotrophs mixed and plated = prototrophs
- Filtered out free DNA = not transformation
- U tube with filter = not transduction
- Hayes = unidrectional transfer by F+ve to F-ve only
Describe the structure of the F plasmid
100kB, tra region for F pilus, oriV for daughter transfer, oriT with insertion elements
Explain the 6 step process of F plasmid conjugation
- F+ randomly contacts F- by F pilus = TraG + TraN mating pore and conjugal bridge
- TraI helicase + TraM + TraD + TraY accessory proteins = relaxosome = bind oriT
- Rolling circle rep = F factor sstrand nicked by TraI helicase @ nic site in oriT
- TraD helps relaxosome recog Tra pore = sstrand thru
- ssDNA -> circularises + dsDNA in recipient
- TraI terminates by recog nic and nicks again
Who discovered Hfr donors?
Hayes and Cavalli Sforza = Lederberg and Tatum cultures were a mix of Hfr and plasmid cells
What are Hfr donors?
- High frequency recombo donors
- F factor integrated into chromosome by homologous recombo of insertion (IS) elements between F plasmid and chromosome
- Hfr = F- bc too big for bridge
How does RecA mediate Hfr donor conjugation and chromosome mapping?
different adjacent markers transferred depending on RecA direction from integration site = chromosome marker mapping
What are the 2 fates of F factor conjugation?
- Conjugation = plasmid no markers
- Conjugation and integration = Hfr donor with markers
What are F’ and who discovered them?
- Separation of Hfr into F factor/F’ and chromosome of the rest
- Haploid donor, diploid recipient for extra bit
- Adelberg and Jacob
How is the F’ excised from the Hfr?
- Homologous recombo between IS elements on F plasmid insertion site and chromosome
- Illegitimate recombo without homology
