Bacterial Conjugation

Instructor: Dr. Amirul Mallick

PRINCIPLE: Transfer of genetic material from one bacteria strain (donor) to

another strain (recipients) is common event that occurs in nature
with the objective of mixing the gene pool , in otherwise
asexually reproducing organisms.
Conjugation is the most widespread process of transferring
genetic materials from one bacteria cell to another .
Unidirectional transfer DNA is mediated and require cell to cell
contact . It was discovered by Lederberg and Tatum In 1946 .
Conjugation is best understood by considering properties of the
F factor , which is a small circular plasmid DNA that can
replicate autonomously in the cell or can integrate in the host
chromosome
When individual cell with an integrated F are isolated an
allowed to form pure colonies , the resulting strain can transfer
chromosomal markers at very high frequency and are termed Hfr
strain. Hfr stands for "high frequency of recombination".

Lederberg & Tatum (1946) Experiment demonstrating

recombination in E. coli. Recombination of 2 complimentary
auxotrophs gives rise to a strain that can synthesize all nutrients.

Bernard Davis experiment demonstrated that physical contact is

required for bacterial recombination.

E. coli has been the model organism in conjugation studies.

Presence of an extrachromosomal genetic element (a plasmid called the F
factor): mediates certain events such as the synthesis of a pilus (the F or sex
pilus), and the rapid infection of all susceptible bacteria with this F factor.
The pilus has been shown to facilitate attachment of the male (or F+ cells) to
the female (or F- cells) which do not contain the F factor.

Conjugation-transfer of the sex factor F:

1.

William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one direction.

2.

Genetic transfer is mediated by sex factor F.

3.

Donor is F+ and recipient is F-.

4.

F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome).

5.

6.

7.

8.

F plasmid contains an origin sequence (O), which initiates DNA transfer. It also contains genes for hairlike cell surface (F-pili or sex-pili), which aid in contact between cells.
No conjugation can occur between cells of the same mating type.
Conjugation begins when the F plasmid is nicked at the origin, and a single strand is transferred using the
rolling circle mechanism.
When transfer is complete, both cells are F+ double-stranded.

Transfer of the F factor

Inoculate 1ml of
overnight donor culture
into 25ml of LB
broth( +tetracycline
30g/ml.

Inoculate overnight
recipient culture into
25ml of LB broth (+
streptomycin 100g/ml
Incubate at 37C in a shaker

Grow recipient and donor culture

upto O.D. reaches 0.8-0.9 at
600nm.
Gently mix 0.2ml each recipient and
donor cultures and incubate in an
incubator at 37C for 90min.
Add 2.0ml of sterile LB into
each tube Incubate the tubes
at 37C for another 90min
Spread plate 0.1ml of
each of the samples
(donor, recipient,
conjugated sample)