Gene Exchange/Gene Mapping Techniques in Bacteria Transformation

Reading List
Microbial genetics. S.R.Maloy, J.E. Cronan & Freifelder
Genetics – a molecular approach. T.A. Brown

Bacterial Transformation – uptake of DNA
This technique was used to first demonstrate that DNA
was the genetic material and not protein.
Griffith in 1928, demonstrated the ‘transformation
principle’
Avery, Macleod & McCarty (1944) later demonstrated
‘transformation’ material was nucleic acid.
Work was done with S. pneumoniae, 2 forms:
normal or ‘S’ form (smooth shiny colonies), when injected into mice, it kills them
mutant or ‘R’ form (rough colonies), it does NOT kill mice when injected into them.

Griffith’s Experiment

Transformation
Begins with the uptake of DNA chromosomal fragments from the surrounding media into cells competent for uptake of DNA.
The donor DNA then undergoes a physical exchange (recombination) and is incorporated into the host cell (e.g. as can be demonstrated by new antibiotic resistance).
Most bacteria are proficient at recombination but very few are competent for uptake of DNA naturally.
Even those that are naturally competent, only a small fraction are competent, but this can be enhanced by culturing under certain conditions.

Natural competence
This results from changes in the bacterial cell wall at a particular stage in the cell cycle (late log phase) and lasts just for a few minutes.
Receptors (between 4-80) on the cell wall are activated by a competence factor.
Competence factors are produced by a limited number of cells, but these then convert the rest of the culture to be competent by expressing 8 to 10 key proteins.
Competent specific proteins are expressed, e.g. autolysin.
Autolysin activates a DNA binding protein and nuclease in the cell wall.

Natural competence

Transformation cont.
Double-stranded DNA (dsDNA) released by bacteria (e.g. during starvation) bind to the ‘competent’ cell surface.
The dsDNA is nicked and one strand is degraded.
The remaining DNA strand then binds to a protein and enters the cell.
The ssDNA if homologous can then recombine into the host genome.

Transformation

Competence ‘for uptake of DNA’
Relatively few bacteria are naturally competent,
best examples are Bacillus subtilus, Streptomyces pneumoniae and Haemophilus influenzae.
However a wide range of prokaryotic and eukaryotic cells can be made competent.

Genetic Mapping by Transformation
a+ b+
b+
a+
Transformation of an
a-b- recipient
If a+b+ are closely linked (physically close), will see a+b+, a+b- and a-b+.
If not closely linked will only see a+b- or a-b+
DNA released & degraded

Bacterial conjugation
Is based on cell to cell contact due to sex-pili encoded
by the F (fertility) plasmid.
Occurs by crossing an F- (female) strain with
an HFr (High Frequency of recombination) due to an F plasmid integrated into the donor chromosome.
2. an F’ (F-prime) strain.

(from Genetics
by Brown)

Integration of F into the chromosome
produces an Hfr of defined position
Hfrs contain the integrated F factor at a defined position in the bacterial chromosome.
Hfrs behave like a big F factor and transfer the chromosome from the point at which the F factor is integrated.
E. coli chromosome is mapped in minutes, these minutes are the time taken to transfer part of the chromosome. The whole chromosome can theoretically be transferred in 100 minutes.
This allows gene mapping experiments.

E. coli chromosome, each arrow denotes a specific Hfr, with point and direction of chromosome transfer
mins
(from Microbial Genetics Maloy et al.)

Genetic Mapping by Conjugation
1. Can the position of a gene by crossing an Hfr x F- and asking if gene is transferred by specific Hfrs
Hfr A
Hfr B
pheA+
Hfr StrS
F- StrR
X
pheA10
Add Hfr to F- at 1:10 leave for 60 mins at 370C.
Plate on minimal media + streptomycin.
Only Hfr B x F- will yield cells (transconjugants) that will grow on minimal media containing streptomycin, pheA maps between the origin of transfer of Hfr A and Hfr B.

Genetic Mapping by Conjugation
2. Time of transfer – interrupted mating (vortex by jigsaw motor)
Hfr A
pheA+
Hfr StrS
F- StrR
X
cysG+
thrA+
pheA10
cysG22
thrA13
Order of transfer into F-
pheA+
cysG+
thrA+
15 min
25 min
35 min

Genetic Mapping by Conjugation
2. Time of transfer – interrupted mating (vortex by jigsaw motor) Mating as described previous but now plate on minimal media + streptomycin + phenyalanine OR cysteine OR threonine
Number of selected StrR
transconjugants
pheA+
cysG+
thrA+
Time of mating interruption