Lecture 6 Flashcards
How does bacteria reproduce ?
- Asexual reproduction via cell division
What is the main mechanism of gaining generic variation in bacteria
- horizontal gene transfer
Vertical gene inheritance in bacteria
What is horizontal gene transfer in bacteria?
- when an organism acquires genes directly from another cell and incorporates them into its genome
- responsible for the spread of fitness enhancing traits, including antibiotic resistance
- provides a mechanism for ongoing adaptive evolution
3 types of horizontal gene transfer
- conjunction
- transduction
- transformation
What is conjunction
- mating
- DNA transfer through cell-to cell contact mediated by a mobile genetic element such as a plasmid
what is transduction
- DNA transfer mediated by a bacteriophage
What is transformation
Uptake of naked DNA into a competent recipient cell
Two different ways conjunction can happen
Diagram on conjunction, transformation and transduction
DNA transfer is _______, from a ____ to ______ cell
Unidirectional
Donor
Recipient
Only _____ of the donor _____ is transferred to the receipt end in genetic exchange
Part
Genome
(Need recombination to insert it?)
What kind of DNA is usually transferred? Can this replicate by itself? What happens to the transferred DNA?
- in most cases the transferred DNA consists of linear fragments that cannot be replicated autonomously
- consequently, for transferred genes to be stably inherited by recipient cells, they must be recombined into the recipient chromosome
- recipient is permanently changed
Why was Escherichia coli the work horse of molecular biology / why was it chosen for study in 1940?
- it is non-pathogenic, rapid growth, simple nutritional requirements
- supports the growth of a range of bacterial viruses - chosen by a group of physicists and biologists (the “phage group”) to study the problem of replication
- carry out genetic crosses, analyse genetic properties
What bacterium was the first that sexual recombination was discovered in?
E.coli
What experiment did Joshua lederberg do? How do you detect genetic change? Why did it not be too slay what dod he do instead ?
- wanted to find out if bacteria recombined their DNA
- Took autotrophic bacteria and crossed them together
- the sensitivity was dependednt on the frequency of reversion - this didn’t show much in terms of usual revertnants vs recombinants.
- need to use doubly marked strains (freq decreased - double reversion - 10-6 X 10-6 = 10-12
- very powerful selection method to detect genetic recombination
I don’t understand this slide
What is an autotroph
- needs nutritional requirements
What is a prototroph
Don’t not need additional factors
Lederburg and tatums experiment
- mixed autographs together and got prototropic colonies which told him there was recombination happening between A- and B-
Why was lederbergs “simple approach” considered so brilliant ?
- It represented the first use of conditional mutants to select against the parental type
- The mutants were double mutants so revelation artefacts were avoided
- The prototrphic recovery technique has enormous sensitivity
- double mutants meant revertants couldn’t happen!!!!
- minted together two populations of bacteria
- ones auxotrphic for two genes and one is auxotrophic for 3 genes
- together they make prorotrophs
The Davis U-tube experiment shows that…
- physical contact is required for genetic recombination
How did the Davis U-tube experiment work?
- took the two strains lederburg had used and put into U-tube separated by a fine filter (molecules in media can move through but bacteria can’t)
- mix for several hours then plate samples from each side of U-tube
- result: no recombinants
Shows physical contact is required between bacteria for recombination
What does conjugation in E.coli K12 depend on?
A plasmid called the F factor
- if a bacteria contains the F plasmid its called (F+)
- if doesn’t F-
Conjugation diagram
- transfer by rolling circle mechanism
What mechanism is used in conjugation when transferring plasmid from F+ to F-
Transfer by rolling circle mechanism
- nicks off and is single stranded then once in the bacteria it circleises and becomes double stranded
F+ and F- bacteria form…
Two F+
Where does transfer in conjugation start?
At the origin of transfer
Key points of conjugation in E.coli K12
F factor can ____ from _____ cell to _____ cell at ____ _____ by _____ ______ mechanism
Transfer
F+
F-
High frequency
Rolling circle
_ becomes _ while _ remains _
F-
F+
F+
F+
_ does not mate with _ due to ____ _____
F+
F+
Surface exclusion
Only F+ and F- can mate
If F plasmids can transfer from cell to cell, how are chromosomal genes transferred by conjugation?
In approximately 1% of cells in a F+ population, F exists not as a plasmid but integrated into the bacterial chromosome
When F is integrated into the chromosome can it transfer chromosomal DNA into the recipient cell? How does this work and what happens to the transferred DNA?
When F is integrated into the chromosome. It can transfer chromosomal DNA to the recipient cell (can make with F- bacteria, origin of transfer peels off)
Conjugation bridge is fragile - the whole chromosome is rarely transferred
The transferred DNA must be incorporated by recombination (two cross overs as the DNA is circular) or it is lost
What did Joshua lederberg observe in relation to the cells with F integrated
- the few cells with F integrated can transfer chromosomal genes at a low but detectable rate
Chromosome transfer vs plasmid transfer
Why do we call it a Hfr strain? How do we get Hfr strains?
- can isolate strains with F integrated
- every celll in the population then transfers chromosomal genes, giving a high frequency of recombination = Hfr strains
When F- is mated with Hfr - chromosome transfer, is it F- or F+
F-
How does HFr formation occur?
Hfr strains are formed by homologous recombination between identical copies of an insertion sequence or transpoon present on F and the chromosome
How manny insertion sequences does the F plasmid have?
1 x IS2
2 x IS3
1 x Tn1000 (transpoon)
How many copies of IS2 and IS3 does the ecoli K12 chromosome have? What does it provide?
- 12 copies of IS2
- 6 copies of IS3
That provide regions of homology with the F plasmid
Diagram showing the change in direction of origin of transfer (look at red arrow)
F can integrate at more then 18 sites in the chromosome as there are several Hfr strains, each with a characteristic origin and direction of transfer
Chromosomal gene transfer by a particular ___ ______ is _______________ from a fixed origin
Hfr strain
Unidirectional
What does the orientation that F inserts into the chromosome depend on?
The orientation of the IS element in the chromosome that it recombines with
F can intergrate at more then __ sites in the chromosome
18
What do the 18 different Hfr strains result form?
F being able to intergrate at more then 18 sites in the chromosome
Each Hfr strain has a characteristic _____ and ______ __ ______
Origin
Direction of transfer
Take home
Recombination into DNA after transfer -viable and non viable
