Prokaryotic Gene Transfer Flashcards
What are the 3 ways bacteria can transfer genetic material and what do these entail?
- Transformation - uptake of naked DNA by competent cells
- Transduction - transport of bacterial DNA by bacteriophages
- Conjugation - temporary direct physical contact
Who discovered transformation and when?
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
What happens in DNA transformtion in nature?
-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
What is competency?
-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
What genera of gram pos and neg bacteria can natural transformation be described?
Gram pos:
Streptococcus
Bacillus
Thermoactinomycetes
Gram neg:
Haemophilus
Neisseria
Moraxella
Acinetobacter
Heliobacter
Pseudomonas
Transformation mechanism in gram pos bacteria - streptococcus pneumoniae?
-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
Transformation mechanism in gram neg bacteria - Haemophilus
-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
Limitations of the transfer, uptake and stabilisation of foreign DNA molecules?
-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
What are 2 methods of artificial transformation?
- Heat shock with CaCl2 treatment of cells
- Electroporation
-usually very high conc of DNA used
-DNA often inserted into plasma
What is bacterial conjugation, who discovered it and when?
-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
Who proved direct cell to cell contact, when and how?
Davis in 1950
-U tube experiment
Look at diagram
-Fine filter excludes passage of bacteria but not small molecules
What do donors contain?
Fertility factor plasmids - F factor
What does the F factor contain?
-100kb long
-tra operon (transfer gene) with genes for pilus formation and gene transfer
What are the 3 types of donor?
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
What is the recipient?
-Designated F-
-Cell that picks up DNA
What happens in F+ X F- mating (conjugation)
-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
Hfr X F- mating (conjugation)
-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-
F’ X F- mating (conjugation)
-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-
What type of conjugation has been used to map the relative location of bacterial genes?
HFr
What does interrupting mating experiments do?
-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
What is transduction?
Transfer of genes by bacterial viruses
What are the 2 possible outcomes of the life cycle of a phage?
- Reproductive-lytic cycle
- Dormancy-lysogenic cycle
What are the steps to the (A) reproductive-lytic cycle and (B) dormancy-lysogenic cycle?
- Adsorption - phage attaches to host cell
- Penetration - phage DNA circularizes and enters lytic or lysogenic cycle
- 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
- Protein assembly - (A) Cell lyses, releasing phage virions (B) Lysogenic bacterium reproduces normally
- Assembly - Occasionally the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle
- Release or back into cycle
What causes (A) Reproductive-lytic cycle and (B) Dormancy-lysogenic cycle?
A - usually caused by a virulemt phage
B - usually temperate phage. bacteria is lysogen. Integrated viral genome = prophage
