L12 - genetics of bacteria 2 Flashcards
how was conjugation discovered?
- mixed two strains of E.coli of different requirements
(met- bio- leu+) (met+ bio+ leu-) and grew on minimal media - colony growth seen meaning bacteria had to be (met+ bio+ leu+)
- bacteria had to be in contact with each other
briefly describe process of conjugation
donor cell transfers genetic info to recipient cell via hollow tube called conjugation pilus (or sex pilus)
what cell forms the conjugation pilus
donor cell
what is the F plasmid
first plasmid to be discovered
carries genes that enable conjugation
features of F plasmid
- origin of replication (oriV)
- origin of transfer (oriT)
- 3x insertion sequences (IS)
- tra genes
what is meant by F+ and F-
F+ = has F plasmid (donor cell) F- = doesn't have F plasmid (recipient cell)
describe process of F plasmid transfer
- donor cell forms conjugation pilus
- relaxosome complex cleaves the F plasmid DNA at oriT then binds to the 5’ end
- relaxosome moves through pilus with single strand of DNA into recipient cell (peels single strand away)
- new complimentary strands synthesised (in donor and recipient)
- ends joined up, there is now F+ in both cells
what forms conjugation pilus?
donor cell - from pilin proteins encoded by tra genes
how long is the process of F plasmid transfer
2 mins
role of relaxosome complex in conjugation of F plasmid
- cleaves F plasmid DNA at oriT and binds to 5’ end
2. moves single strand of DNA through pilus to recipient cell
name of proteins that form the conjugation pilus
pilin proteins
what is the single strand of F plasmid DNA that is transferred called?
T strand
what is Hfr strain of E.coli?
E.coli that contain F plasmid integrated into genome
high frequency of recombination
what is the importance of the IS sequence?
it is where homologous recombination occurs to integrate the F plasmid into the bacterial genome
describe conjugation of Hfr strains
- Hfr strain can conjugate with an F- recipient cell
- when the integrated F plasmid transfers (is peeled) from the donor genome it may take small regions of bacterial DNA with it (it is peeled, the whole F plasmid doesn’t excise)
- these regions of bacterial DNA could be transferred and integrate into the recipient genome with the F plasmid
how long would it take to transfer the entire E.coli genome to recipient cell? does this happen often? why/why not
100 mins
no
conj pilus usually breaks down before then
after Hfr conjugation, is the recipient cell F+?
no , the conjugation pilus breaks down before the entire transfer of the F plasmid
use of interrupted conjugation (mating)
map E.coli genome
how does interrupted mating produce a genetic map
the earlier a gene is transferred to the F plasmid, the closer it is to the integrated F plasmid
what E.coli strains are used in interrupted mating
Hfr :
F-
how can you see if a gene has transferred to the F- plasmid?
grow it on selective media for the gene
describe process of interrupted mating
Hfr strain will be sensitive to an antibiotic but + for all other genes - that are trying to be transferred
- mix Hfr and F-
- at 5, 10 , 15 , 20 , 25 mins take sample, vortex and plate on different selective media for all of the genes of interest
- count colonies and plot graph
what does the plateu of an interrupted mating graph show
the breakage of the conj pilus - no. of colonies wont just keep increasing because eventually it breaks
what genes are first to transfer if the F plasmid integrates into IS1
leu / val
depending on orientation of ori of transfer
what genes are first to transfer if the F plasmid integrates into IS2
leu / thr
depending on orientation of ori of transfer
features to note about naturally occurring plasmids
- not all can transfer between bacteria
- not all can integrate into bacterial genome
describe features of R plasmids
- have tra genes so can conjugate
- carry genes that confer antibiotic resistance
describe features of ColE 1
- found in E.coli
- no tra genes (cant conjugate)
- produces the protein colicin which is toxic to other bacteria
describe features of Ti plasmids
- in soil bacteria
- has tra genes
- can also transfer their T region into plant cells - integration into plant chromosome causes crown gall disease
what causes crown gall disease in plants?
integration of T region of Ti plasmid of soil bacteria into the plant chromosome
