Lecture 2: Prokaryotic Genetics II

Question 1

Explain the exchange of genetic material :

Animal vs Bacteria

Answer 1

In eukaryotes, genetic exchange occurs during meiosis and mitosis via genetic recombination.

Bacterial processes are not so regular; however, they serve the same aim: to mix the genes from two different organisms together.

Question 2

There are 3 types of processes that GENETIC EXCHANGE in bacteria LIST AND EXPLAIN THEM

Answer 2

1. CONJUGATION: direct transfer of DNA from one bacterial cell to another
2. TRANSDUCTION: use of a bacteriophage to transfer DNA between cells.
3. TRANSFORMATION: naked DNA is taken up from the environment by bacteria

Question 3

How was the Conjugation process identified and termed? = 4

Answer 3

1. In 1946 Lederberg and Tatum showed that bacteria can transfer andf recombine Genetic Material.
2. Bernard Davis showed that CONTACT between the bacterial strains was required for this genetic exchange.
3. U-shaped tube Experiment.
4. The direct contact process of gene transfer was termed CONJUGATION.

Question 4

Look at the Conjugation U-shaped tube experiment diagrams.

Answer 4

Look at the Conjugation U-shaped tube experiment diagrams.

Question 5

Is Conjugation Reciprocal?

Answer 5

In 1953, William Hayes determined that conjugation in E.coli is NOT RECIPROCAL,

i.e. a donor cell transfers part of its genome to another (recipient) cell. Donors have the fertility factor (F), designated F+, strains which lack F are recipients (F-).

Question 6

Where does Conjugation begin and How? = 3

Answer 6

1. F gene is on the episome plasmid (F factor) - contains an
   - Ori (origin of replication)
   - Genes required for conjugation eg the pili (extension of cell membrane) which makes contact with a receptor on F- cell and pulls the cells together.
2. Conjugation only occurs between F+ and F- cells.
   - usually, the only genes exchanged are on the F factor.
3. Transfer is initiated by nicking a strand on F (at origin, oriT), which separates from the plasmid and moves into the recipient cell.

Question 7

Explain Conjugation and F factor relationship. = 3

Answer 7

1. The F factor in the cell makes a single-stranded copy of itself = rolling circle replication.
2. The SS copy is cast out through a pore into the recipient cell where the 2nd strand is made to give DS DNA.
3. So the copy of F remains in the donor and now the recipient cell also has F.

Question 8

Episome and F (fertility) factor

Answer 8

Episome = plasmid which can freely REPLICATE AND INTEGRATE into the bacterial chromosome.

F (fertility) factor - an E.coli episome that regulates transfer, replication, and insertion.

Question 9

What does conjugation explain? 2

Answer 9

1. Conjugation ONLY explains TRANSFER OF F GENES, not chromosomal genes (which was also observed by Lederberg and Tatum)
2. Hfr (high frequency) strains have the F factor integrated into the bacterial chromosome.

• Hfr strains can also under conjugation with F-

Question 10

What happens if Hfr and F- conjugation occurs?

7

Answer 10

1. If conjugation occurs between Hfr and F-, then the chromosome follows the F factor into the recipient cell…
2. in conjugation, F is nicked and the 5’ end moves into the F- cell.
3. the amount of transfer depends on the time the cells are joined
4. The transferred strand replicates …
5. In the recipient cell, can get crossed over with the inserted DNA.
   … and crossing over takes place between the donated Hfr chromosome and the original chromosome of the F- cell.
6. Crossing over may lead to the recombination of alleles
7. The linear chromosome is degraded.

Question 11

Explanation of F- Cell Conversion and F Plasmid Integration.

Answer 11

1 * F- cell virtually never converted to an F+ or HFr (when mate with a HFr cell), as the F factor is nicked in the middle when the transfer is initiated.

2 * To become F+ the entire chromosome must be transferred, which hardly ever happens, as it would mean the conjugating cells staying together for a long time.

3 * Hfr cells produced via F plasmid integration only occur in 1/10000 cells.

4 * F factor is excised from chromosomes at a low rate

Question 12

Explanation of F’ Cells and Sexduction: what is sexduction, 6 Steps

Answer 12

F’ cells contain the F factor plus some bacterial genes that were transferred with it when the F factor is excised from an Hfr cell’s chromosome…This is called sexduction

1. Crossing over takes place within the Hfr chromosome.
2. When the F factor excises from the bacterial chromosome, it may carry some bacterial genes (in this case, lac) with it.
3. F’ cells can conjugate with F- cells
4. During conjugation, the F factor with the lac gene is transferred to the F- cell, …
5. …producing a partial diploid with two copies of the lac gene.

6.Excision is uncommon, about 1 in 10000 cells, but does happen to get F’ cells.

Question 13

what are merozygotes

Answer 13

4. Partial diploids = merozygotes

• a bacterial cell having a second copy of a particular chromosomal region in the form of an exogenote.

A partially diploid Escherichia coli cell formed from a complete chromosome (the endogenote) plus a fragment (the exogenote).

Question 13

what are merozygotes

Answer 13

4. Partial diploids = merozygotes

• a bacterial cell having a second copy of a particular chromosomal region in the form of an exogenote.

A partially diploid Escherichia coli cell formed from a complete chromosome (the endogenote) plus a fragment (the exogenote).

Question 14

How to map bacterial genes using conjugation?

Answer 14

1. Conjugation can be used to map bacterial chromosome genes by interrupted conjugation. The transfer of entire E. coli chromosome in Hfr = 100 min.

2 * Transfer starts with F factor and proceeds in one direction

3 * If transfer is interrupted, then only parts are transferred to the recipient. so relative distances of genes on the chromosome can be measured across time.

azi – sodium azide (S/R) ton – T1 phage (S/R)

Question 15

Explain interrupted Conjugation Mapping (3).

When is this technique used?

Answer 15

1. Chromosome transfer from the Hfr into the F- is slow: about 100 minutes to transfer entire chromosome.

2 * The conjugation process can be interrupted by agitation (using a kitchen blender).

3 * By interrupting the mating at various times, can determine the proportion of F- cells that have received a given marker.

This technique can be used to make a map of the circular bacterial chromosome.

Question 16

F factor orientation determines the direction of gene transfer, and site & orientation differ between Hfr strains - Explain this process (6).

Answer 16

F factor orientation determines the direction of gene transfer, site & orientation differ between Hfr strains

1. Transfer always begins within F, and the orientation of the F determines the direction of transfer.
2. In Hfr1, F is integrated between the ‘leu’ and ‘azi’ genes;…
3. …so the genes are transferred beginning with ‘leu’.
4. In Hfr5, F is integrated between ‘thi’ and ‘his’.
5. F has the opposite orientation in this chromosome; so the genes are transferred beginning with ‘thi’.
6. Still get the same relative distances between genes and the same order

**First evidence that bacterial chromosome is circular

Question 17

Characteristics of E.coli cells with different types of F factor.
(type, F Factor Characteristics, Role in Conjunction)

Answer 17

Type F+
- Present as separate circular DNA
- Donor

Type F-
- Absent
- Recipient

Type Hfr
- Present, integrated into bacterial chromosome
- High-frequency donor

Type F’
- Present as separate circular DNA, carrying some bacterial genes
- Donor

Question 18

Results of conjugation between cells with different F factors.

Conjugating - Cell Types Present after Conjugation

Answer 18

Conjugating ——— Cell Types Present after Conjugation

1. F+ x F- 2 F+ cells (F- cell becomes F+)
2. Hfr x F- one Hfr cell and one F- (no change)*
3. F’ x F- two F’ cells (F- cell becomes F’)

*Rarely, the F- cell becomes F+ in an Hfr x F- conjugation if the entire chromosome is transferred during conjugation.

R plasmids confer antibiotic resistance and can also be transferred by conjugation.

Question 19

Conjugation between an F+ and F− cell usually results in:

Answer 19

two F+ cells.

Question 20

Mapping bacterial genes can be done by?:

Answer 20

Interrupted Conjugation…
Distance between genes are measured by the time required for DNA transfer from Hfr cells to F– cells

Question 21

Explain Bacteriophage (bacterial viruses) = 5

Answer 21

1. Viruses can infect all organisms. Comprise protein coat and internal nucleic acid.
2. Bacterial viruses = bacteriophage or phage.
3. Protein coat binds to the bacterial surface, then injects the phage Nuclei Acid.

4 * Phage can only reproduce inside the host cell.

5. When you plate bacteria infected with phage, will see cell death resulting in plaques on the lawn of bacteria…

A virus consists of a protein coat… surrounding a piece of nucleic acid - in this case, DNA.

Question 22

Phage “Life” Cycles: Lytic

Answer 22

Lytic Cycle:
1 - Phage infects the host bacterial cell

2 - Phage DNA takes over the host cell’s machinery

3 - The host cell’s resources are used to produce new phages

4 - Host cell lyses (bursts open) and releases the newly formed phages

5 - These phages can go on to infect other bacterial cells

Question 23

Phage “Life” Cycles: Lysogenic Cycle

Answer 23

Lysogenic Cycle:

1 - Phage infects the host bacterial cell

2 - Phage DNA integrates into the host cell’s chromosome (becoming a prophage)

3 - The prophage is replicated along with the host DNA during cell division

4 - The host cell and its descendants carry the integrated phage DNA in their genomes

5 - Under certain conditions, the prophage may exit the host chromosome and initiate the lytic cycle, leading to the production of new phages and lysis of the host cell

Key Point:
The lytic cycle results in the immediate destruction (lysis) of the host cell, while the lysogenic cycle involves the integration of phage DNA into the host genome and can lead to the transmission of phage DNA to future generations of host cells.

Question 24

Explain Transduction: What are the 2 forms and explain them.

Answer 24

Process of moving bacterial DNA from one cell to another using phage. Two forms of transduction: 1. generalised: any piece of the bacterial genome can be transferred 2. specialised: only specific pieces of the chromosome can be transferred Most bacteriophages have a limited host infection range, so transduction is normally between bacteria of the same or closely-related species.

Question 25

Explain Generalised Transduction: 3

Answer 25

1. 1952 – Lederberg and Zinder: identified genetic exchange without any cell-cell contact? 2. Genetic exchange did NOT take place through conjugation. 3. A phage was the agent of transfer = transducing phage, and the recombinant bacteria are transductants. The process? 1. Two auxotrophic strains of 'salmonella typhimurium' were mixed... 2. ...and plated on minimal medium. 3. Some prototrophic colonies were obtained. 4. When the two strains were placed in a Davis U-tube,... 5. ...which separated the strains by a filter with pores too small for the bacteria to pass through,... 6. prototrophic colonies were obtained from only one side of the tube.

Question 26

explain Generalised Transduction in LYTIC CYCLE: What is the process?

Answer 26

- In lytic cycle, bacterial chromosome broken into fragments and some phage will take up a piece of this DNA into phage coat. - The phage then infects another cell and by double crossover the genes integrate into the genome . The process = 1. Baceria are infected with phage 2. The bacterial chromosome is fragmented... 3. and some of the bacterial genes become incorporated into a few phages. 4. Cell lysis releases transducing phages. 5. If the phage transfers bacterial genes to another bacterium, recombination may take place and produce a transduced bacterial cell.

Question 27

Generalised Transduction: Transduction is rare because it requires: 3 (and why?)

Answer 27

Transduction is rare (1/100,000 to 1/1,000,000) because it requires: 1. phage degrades the bacterial chromosome 2. phage packages this bacterial DNA 3. bacterial genes need to recombine with the recipient chromosome * Due to small phage size, only ±1% of bacterial chromosomes can be transduced, thus only genes close together on chromosome transferred together = co-transduced. - Very unlikely to get two separate transductions together. - So, the rate of co-transduction indicates close physical distances between bacterial genes * Not all phage can transduct

Question 28

Generalised Transduction to Map Genes: the process?

Answer 28

1. A donor strain of bacteria that is a+ b+ c+ is infected with phage. 2. The bacterial chromosome is broken down, and bacterial genes are incorporated into some of the progeny phages,... 3. ...which are used to infect a recipient strain of bacteria that is a- b- c-. 4. Transfer of genes from the donor strain and recombination produce transductants in the recipient bacteria. CONCLUSION: Genes located close to one another are more likely to be contransduced; so the rate of cotransduction is inversely proportional to the distance between genes.

Question 29

Explain Specialised Transduction:

Answer 29

1. Only genes near particular sites on chromosomes are transferred, which requires lysogenic bacteria. 2. When prophage is excised, it takes some bacterial chromosomes with it, which can then be injected into another bacterium ... similar to F’ cells where F plasmid carries genes from one cell to another

Question 30

Explain Transformation: Explain the process = 4

Answer 30

Requires the uptake of DNA from outside the cell and its integration into the bacterial chromosome (or plasmid). Process: 1. One strand of the DNA fragment enters the cell; the other is hydrolyzed 2. the single-stranded fragment pairs with the bacterial chromosome and recombination takes place. 3. The remainder of the single-stranded DNA fragment is degraded. 4. When the cell replicates and divides, one of the resulting cells is transformed and the other is not.

Question 31

Explain the particular characteristics of Transformation:

Answer 31

1 * Competent bacterial cells can take up DNA through their cell membrane. 2. Level of competence differs according to: * species * growth stage of bacteria * [DNA] in the environment * environmental factors 3 * Virtually any type of DNA (not just bacterial) can be taken up. When the DNA enters the cell one strand is broken up, while the other strand moves across the membrane where it can be integrated by homologous recombination into the chromosome. 4 * Any remaining Single Stranded SS DNA is degraded. 5 * In the lab, the transformation of Double-Stranded DNA plasmid is often carried out to amplify cloned genes in bacteria

Question 32

Transformation in the Laboratory: Chemical transformation vs Electroporation.

Answer 32

- Used in laboratory to introduce DNA into bacteria. Strains developed which are more competent. Chemical transformation: Treatment with CaCl2, heat or electrical field increases the permeability of the cell membrane to enhance DNA uptake. Enable usually incompetent cells (such as E. coli) to be transformed. Electroporation: electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing DNA to be introduced into the cell.

Question 33

Transformation Mapping: What does it require? Method? Process: 4

Answer 33

1. Requires 2 strains which differ in several traits, donor and recipient. 2. Method: - Purify and fragment donor DNA, treat the recipient to make competent, - then add DNA (the recipient is thus the transformant). - Genes are mapped by determining the rate 2 or more genes are cotransformed... PROCESS: 1. DNA from a donor cell is fragmented 2. Fragments are taken up by the recipient cell. 3. After entering the cell, the donor DNA becomes incorporated into the bacterial chromosome through crossing over. 4. Genes that are close to one another on the chromosome are more likely to be present on the same DNA fragment and be recombined together. CONCLUSION: The rate of biotransformation is inversely proportional to the distances between genes.

Question 34

WHY is Transformation used in Labs?

Answer 34

1. Scientists use bacterial transformation to introduce genes of interest and make multiple copies of these quickly and inexpensively. 2. Plasmids have been modified to enable insertion of genes in specific positions using restriction enzymes and DNA ligase (multiple cloning sites) Lab class: Transform E. coli and look to see if a protein encoded by a gene of interest (GFP) is expressed.

Question 35

Transformation and Plasmids uses?

Answer 35

Plasmids have been modified to enable the insertion of genes in specific positions using restriction enzymes and DNA ligase (multiple cloning sites)/ recombination