Test1: Lect6 Peter Zuber Flashcards
Describe the initial experiment that showed that transformation was possible:
1: Strept. pneumoniae has two forms, R (no capsule) and S (capsule).
2: when R is gained, S will not come back
3: Inject mice with heat killed S, and live R
4: only S is pathogenic
5: R becomes S (must have taken material from S)
6: mice die, S is isolated from the dead mice, no R
How did people know that DNA was what was being used for transformation?
Tried experiment without adding proteins, lipids, and ribonucleotides, and it still transferred (R became S)
- If DNA destroyed from S mouse lives, no DNA recovered
People did not believe that DNA was being transferred, they assumed a protein contaminant.
How was this proven to not be the case?
Shown that DNA was hereditary material in the 1950s, this overcame the objection.
Another objection to transformation: Yes, DNA was the transforming principle, but it acted directly on capsule synthesis.
Counter?
Extremely Rough (ER) can go to R -> S -> ER. Shown Streptomycin-resistant Pneumococcus can transfer resistance. Not isolated case
How is DNA taken into the cell in transformation?
How do we know?
Receptors
Because high levels of DNA saturate receptors and uptake is plateaued. Addition of competitor adjusts plateau.
When in the bacterial cell cycle is transformation optimized?
Right at the end of replication
How does quorum sensing effect transformation rates?
- Describe the mechanism:
It increases it - Describe the mechanism: Two component regulatory system. ComX (diffusable pheromone) is detected by ComP transmembrane protein. Phosphorylated. -> ComP-P phosphorylates ComA -> ComA - P -> ComA - P activates ComQ which facilitates entry of the gene
How does the DNA enter the cell in transformation?
There is a transmembrane helicase, which brings one strand of the DNA into the cell.
RecA forms a filament with it.
What does recA do?
It forms a filament with with ssDNA, fascilitates the replacement of a chromosomal strand with ssDNA.
Auxotroph:
A mutant organism (typically a bacterium or fungus) that requires a particular additional nutrient that the normal strain does not.
- Cannot grow in minimal medium but can in complete medium
Prototroph:
A microorganism that has the same nutritional requirements as the parent organism.
- Can grow in minimal medium (ammonia, sulfate, and carbon source)
In the discovery of conjugation, Lederburg struggled to tell whether gene transfer or reversion to prototype mutations were occurring for his autotrophs. How did he fix this?
- Process?
He started using multiple auxotrophic markers. Reversion at one auxotrophic marker may occur, but the odds are small that both auxotrophic markers will become prototrophic.
- Process:
1: two strains, different growth requirments
2: mix, allow growth overnight
3: collect cells by centrigution
4: plate on minimal media (recombinants will grow)
5: control for reversion by colony formation from each strain alone (cells never mixed)
How was it discovered that conjugation was not transformation?
Cells could not get genes from lysate of other cells.
No diffusible component, contact necessary.
What would we see if there was linkage vs whole genome vs genes exchange randomly.
- Which is it?
1: Linkage would result in certain genes being transferred more often with others.
2: Whole genome all or none
3: Random, equal probability of any gene.
- Which is it?
Linkage, F factor necessary.
F factor:
- Define:
- Two origins?
- IS elements?
- Define:
Plasmid, transferred 1:10 cells by rolling replication in pilli connection. - Two origins?
OriT: for Origin transfer, rolling circle replication
OriV: for normal replication of plasmid in host - IS elements?
Insertion Sequence genes, they allow transposition (allow inserting into different genes)
How was weight of F+ factor determined?
Transferred from F+ E. coli to F- S. Marcescens
E coli has different base comp. = different mass.
CeCl centrifugation separated E coli F+ from the S. Marc.
Found 100 kB sequence
Hfr:
- Stands for:
- What is it?
- Stands for:
High frequency recombination - What is it?
The F plasmid incorporated into the bacterial genome
Explain how gene transfer occurs using Hfr?
1: The F plasmid begins rolling circle replication from its origin, which is on the e coli genome, since it has incorporated into the genome.
2: It will transfer genes in order as it travels unidirectionally across the genome.
How can space be measured on the genome using Hfr?
1: Use a waring blendor to separate conjugated cells, which stops genetic transfer.
2: Stop at 1 minute, 2 minute, 5, 10, 15 etc.
3: You can see which genes have had time to make it through, the longer they take, the farther they are from the origin of rolling replication
4: KEEP IN MIND THAT DUE TO SPONTANEOUS ATTACHMENT THE TIME GROW EXPONENTIALLY IN PROPORTION TO DISTANCE
If I have gene 1 at 5 minutes, and gene 2 at 10 minutes? Is gene 2 twice as far from the origin as gene 1?
NO! It is more likely to be 1.2 or 1.4 times as far from the origin! Time grows exponentially in proportion to actual distance.
I see no recombination past a certain gene in a Hfr. Why?
After that gene is a lambda virus in its lysogenic state. If it is transferred into a new cell, that cell will not have C1 to repress lytic expression, and it will lyse and kill any cell it is transferred into.
How was it tested that Hfr was a random event which could be performed by only some F+ carriers?
F+ divided into 50 small samples and 1 large sample. 50 samples from bulk culture compared to 50 samples from small culture. Much smaller variance in bulk culture shows the event is random.
Why does Hfr transfer of genes not make cell F+?
Because the origin of rolling replication is in the middle of the Hfr plasmid. So to transfer the whole F plasmid genome, you would need to transfer ALL of E-coli’s genome (half of F is transferred first, half transferred last)
What are the possibilities for F+ conjugation?
1A: crossing over, entry into genome 1B: F plasmid stays vector 2: conjugation 3A: transfer of some e-coli genes, but no F plasmid 3B: transfer of F+ plasmid, but no genes
Three factor cross: Two strands Leu+ Arg+ Met+ (inserted F plasmid ssDNA) Leu- Arg- Met- (chromosomal) Select for Leu+ - Name all possible recombinants, showing were crossing over necessary: - Which will be the least to occur? - Why?
- Name all possible recombinants, showing were crossing over necessary: Note: leu+ must be present, that is what your selecting for. X Leu+ X Arg+ Met+ X Leu- X Arg- Met- -> Leu+ Arg- Met- \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ X Leu+ Arg+ X Met+ X Leu- Arg- X Met- -> Leu+ Arg+ Met- \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ X Leu+ Arg+ Met+ X X Leu- Arg- Met- X -> Leu+ Arg+ Met+ \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ X Leu+ X Arg+ X Met+ X X Leu- X Arg- X Met- X -> Leu+ Arg- Met+ - Which will be the least to occur? Leu+ Arg- Met+ - Why? Requires 4 crossing over events (quadruple events), others require two.
F’ plasmid:
A F plasmid with a chromosomal gene in it
If I want a specific F’ plasmid, how would I select for it?
You want them to be RecA-, so they cannot catalyze crossing over of plasmid back into bacterial genome.
What is an ICE?
- What do ICEs do?
Integrative and conjugative element.
- What do ICEs do?
Carry genes for conjugative transfer, as well as genes for their own removal genome and insertion (into a specific site) into other genomes. Transferred by bacterial conjugation.
An experiment was performed confirming horizontal transfer of ICE, how was it set up?
- What did they learn?
Donor: ICEBs1::lacO, produces mCherry
Recipient: LacI-GFP, ICE-
Conjugation provides the LacO site for O to bind and fluoresce.
- What did they learn?
Bacteria transfer down a single chain of bacteria
ICEs proven not to be replication by?
conG+ genome ICE has conG-, can only transfer once. This is what we see. Not replication, conjugation.
