Test1: Lect6 Peter Zuber

Question 1

Describe the initial experiment that showed that transformation was possible:

Answer 1

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

Question 2

How did people know that DNA was what was being used for transformation?

Answer 2

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

Question 3

People did not believe that DNA was being transferred, they assumed a protein contaminant.
How was this proven to not be the case?

Answer 3

Shown that DNA was hereditary material in the 1950s, this overcame the objection.

Question 4

Another objection to transformation: Yes, DNA was the transforming principle, but it acted directly on capsule synthesis.
Counter?

Answer 4

Extremely Rough (ER) can go to R -> S -> ER.
Shown Streptomycin-resistant Pneumococcus can transfer resistance.
Not isolated case

Question 5

How is DNA taken into the cell in transformation?

How do we know?

Answer 5

Receptors

Because high levels of DNA saturate receptors and uptake is plateaued. Addition of competitor adjusts plateau.

Question 6

When in the bacterial cell cycle is transformation optimized?

Answer 6

Right at the end of replication

Question 7

How does quorum sensing effect transformation rates?

- Describe the mechanism:

Answer 7

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

Question 8

How does the DNA enter the cell in transformation?

Answer 8

There is a transmembrane helicase, which brings one strand of the DNA into the cell.
RecA forms a filament with it.

Question 9

What does recA do?

Answer 9

It forms a filament with with ssDNA, fascilitates the replacement of a chromosomal strand with ssDNA.

Question 10

Auxotroph:

Answer 10

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

Question 11

Prototroph:

Answer 11

A microorganism that has the same nutritional requirements as the parent organism.
- Can grow in minimal medium (ammonia, sulfate, and carbon source)

Question 12

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?

Answer 12

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)

Question 13

How was it discovered that conjugation was not transformation?

Answer 13

Cells could not get genes from lysate of other cells.

No diffusible component, contact necessary.

Question 14

What would we see if there was linkage vs whole genome vs genes exchange randomly.
- Which is it?

Answer 14

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.

Question 15

F factor:

• Define:
• Two origins?
• IS elements?

Answer 15

• 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)

Question 16

How was weight of F+ factor determined?

Answer 16

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

Question 17

Hfr:

• Stands for:
• What is it?

Answer 17

• Stands for:
  High frequency recombination
• What is it?
  The F plasmid incorporated into the bacterial genome

Question 18

Explain how gene transfer occurs using Hfr?

Answer 18

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.

Question 19

How can space be measured on the genome using Hfr?

Answer 19

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

Question 20

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?

Answer 20

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.

Question 21

I see no recombination past a certain gene in a Hfr. Why?

Answer 21

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.

Question 22

How was it tested that Hfr was a random event which could be performed by only some F+ carriers?

Answer 22

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.

Question 23

Why does Hfr transfer of genes not make cell F+?

Answer 23

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)

Question 24

What are the possibilities for F+ conjugation?

Answer 24

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

Question 25

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?

Answer 25

- 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.

Question 26

F’ plasmid:

Answer 26

A F plasmid with a chromosomal gene in it

Question 27

If I want a specific F’ plasmid, how would I select for it?

Answer 27

You want them to be RecA-, so they cannot catalyze crossing over of plasmid back into bacterial genome.

Question 28

What is an ICE?

- What do ICEs do?

Answer 28

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.

Question 29

An experiment was performed confirming horizontal transfer of ICE, how was it set up?
- What did they learn?

Answer 29

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

Question 30

ICEs proven not to be replication by?

Answer 30

conG+ genome ICE has conG-, can only transfer once. This is what we see. Not replication, conjugation.