Prokaryotic Gene Transfer

Question 1

What are the 3 ways bacteria can transfer genetic material and what do these entail?

Answer 1

1. Transformation - uptake of naked DNA by competent cells
2. Transduction - transport of bacterial DNA by bacteriophages
3. Conjugation - temporary direct physical contact

Question 2

Who discovered transformation and when?

Answer 2

Fred Griffith in 1928
S strain (kills mouse) and R strain (doesnt kill mouse), heat killed S cells (doesnt kill mouse), heat killed S strain and R strain (kills mouse)-living S cells in blood sample from dead mouse

Question 3

What happens in DNA transformtion in nature?

Answer 3

-Occurs when bacteria lyse and release DNA
-Fragments can be large and contain several genes
-Can be bound by a competent cell and taken inside

Question 4

What is competency?

Answer 4

-Ability to uptake DNA
-Complex phenomenon depending on several factors
-Bacteria need to be in a certain stage of growth e.g streptococcus pneumoniae in exponential phase secretes a small protein called competence factor that stimulates 8-10 new proteins

Question 5

What genera of gram pos and neg bacteria can natural transformation be described?

Answer 5

Gram pos:
Streptococcus
Bacillus
Thermoactinomycetes

Gram neg:
Haemophilus
Neisseria
Moraxella
Acinetobacter
Heliobacter
Pseudomonas

Question 6

Transformation mechanism in gram pos bacteria - streptococcus pneumoniae?

Answer 6

-A competent cell binds a ds DNA molecule
-The DNA is cleaved by an endonuclease to fragments 5 to 15kb
-One strand is hydrolysed by an exonuclease
-Other strand moves through the plasma membrane, can align to homologous region and integrate by recombination

Question 7

Transformation mechanism in gram neg bacteria - Haemophilus

Answer 7

-Does not produce competence factor
-Takes up DNA only from closely related species
-ds DNA complexed w protein is taken inside membrane vesicles
-Specificity occurs due to a special 11bp sequence that is repeated higher than 1400 in haemophilus influenza DNA
-Transferred DNA must have this sequence

Question 8

Limitations of the transfer, uptake and stabilisation of foreign DNA molecules?

Answer 8

-limited release and stability of adaptive DNA in the environment
-limits on competence development
-limits on host range of transfer and maintenance mechanism of mobile genetic elements
-recipient restriction enzyme activity
-limited ability of foreign DNA to integrate into a replicating genetic element due to a lack of DNA sequence similarity

Question 9

What are 2 methods of artificial transformation?

Answer 9

1. Heat shock with CaCl2 treatment of cells
2. Electroporation

-usually very high conc of DNA used
-DNA often inserted into plasma

Question 10

What is bacterial conjugation, who discovered it and when?

Answer 10

-Transfer of DNA by direct cell to cell contact
-For conjugation, a donor and recipient must come into direct contact
-Discovered by Lederberg and Tatum in 1946
-same exp we carried out w the diff minimal media plates that lack what each need to grow but if you plate them together they grow
-Not enough to prove cell to cell contact

Question 11

Who proved direct cell to cell contact, when and how?

Answer 11

Davis in 1950
-U tube experiment
Look at diagram
-Fine filter excludes passage of bacteria but not small molecules

Question 12

What do donors contain?

Answer 12

Fertility factor plasmids - F factor

Question 13

What does the F factor contain?

Answer 13

-100kb long
-tra operon (transfer gene) with genes for pilus formation and gene transfer

Question 14

What are the 3 types of donor?

Answer 14

1) F+ - F factor alone autonomous -free in cytoplasm
2) F’ - Factor autonomous with additional genes e.g for antibiotic resistence etc
3) HFr -integrated F factor plasmid -in chromosome

Question 15

What is the recipient?

Answer 15

-Designated F-
-Cell that picks up DNA

Question 16

What happens in F+ X F- mating (conjugation)

Answer 16

-A pilus forms between 2 cells, extending from the F+ cell
-Single strand of F factor is moved across pilus
-Each single strand is replicated
-F- becomes F+ due to acquisition of F factor plasmid- donor stays F+ plasmid

Question 17

Hfr X F- mating (conjugation)

Answer 17

-Integrated F plasmid with functional tra operon
-HFr: high frequency of recombination because a very high efficiency of chromosomal gene transfer when compared with F+
-DNA transfer origin begins with F factor in middle of plasmid
-While replicating some chromosome moves through pilus
-Only part of F factor is transferred therefore F- does not become donor

-recipient is changed but doesnt have all functionality of F plasmid so not a donor - makes recombinant F-

Question 18

F’ X F- mating (conjugation)

Answer 18

-F’ occurs due to imprecise excision of integrated F plasmid from chromosome
-autonomous but plasmid may have additional genes to tra operon genes e.g antibiotic resistence - plasmid is bigger as has more genes
-Mating virtually identical to F+ X F-

Question 19

What type of conjugation has been used to map the relative location of bacterial genes?

Answer 19

HFr

Question 20

What does interrupting mating experiments do?

Answer 20

-Hfr conjugation used to map relative loaction of bacterial genes
-Observed that the chromosome moves from donor to recipient at constant rate
-In an interrupted mating experiment, the conjugation bridge is broken at various intervals after the start of conjugation by mixing the culture vigourously
-The order and timing of the gene transfer can be determined because they are in direct reflection of genes on the bacterial chromosomes - first genes that make it across are closer to the plasmid

Question 21

What is transduction?

Answer 21

Transfer of genes by bacterial viruses

Question 22

What are the 2 possible outcomes of the life cycle of a phage?

Answer 22

1. Reproductive-lytic cycle
2. Dormancy-lysogenic cycle

Question 23

What are the steps to the (A) reproductive-lytic cycle and (B) dormancy-lysogenic cycle?

Answer 23

1. Adsorption - phage attaches to host cell
2. Penetration - phage DNA circularizes and enters lytic or lysogenic cycle
3. DNA synthesis - In lytic cyle (A) -new phage DNA and proteins are synthesised and assembled into virions, -In lysogenic cycle (B) -Phage DNA integrates within bacterial chromosome by recombination becoming a prophage
4. Protein assembly - (A) Cell lyses, releasing phage virions (B) Lysogenic bacterium reproduces normally
5. Assembly - Occasionally the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle
6. Release or back into cycle

Question 24

What causes (A) Reproductive-lytic cycle and (B) Dormancy-lysogenic cycle?

Answer 24

A - usually caused by a virulemt phage

B - usually temperate phage. bacteria is lysogen. Integrated viral genome = prophage

Question 25

Where are bacterial genes found?

Answer 25

Incorporated into a phage capsid due to errors in the virus life cycle
These ‘defective’ viruses then inject them into another bacterium completing transfer

Question 26

What are the 2 types of transduction?

Answer 26

1. Generalised transduction occurs as a result of lytic cycle and any random segment of DNA can be transferred
2. Specialised transduction occurs as a result of lysogenic cycle and only genes adjacent to integrated virus are transferred

Question 27

What happens in generalised transduction?

Answer 27

1. Occasionally during maturation, a bacteriophage head or capsid assembles around a fragment of the donor becaterium’s degraded DNA
2. The ‘defective’ bacteriophages are released
3. The bacteriophage carrying the donor bacterium’s DNA absorbs to a recipient bacteria
4. Upon infection the bacteriophage delivers the donor bacterium’s DNA it is carrying into the recipient bacterium

Question 28

What happens in specialised transduction?

Answer 28

1. Occasionally during spontaneous excision, a small piece of the donor bacterium’s DNA adjacent to the bacteriophage insertion site is picked up - The bacterial DNA incorporated in the phage capsid replaces some of the phage genome
2. These bacteriophages adsorb to a recipient bacterium and inject its genome
3. The bacteriophage carrying the donor bacterial DNA inserts into the recipient bacterium’s nucleoid