organization and integration of F plamid into bacterial strain through conjugation

1. Organization of the F
Plasmid
Dr. K. Madhuri
Dept. of Biotechnology
Mahatma Gandhi University
Nalgonda

3. Organization of the F Plasmid:
The F plasmid is a double-stranded circular DNA
molecule of about 100 kpb (105 nucleotide pairs). It is
about 30 pm long which is about 1/40 of the length of
the bacterial (E. coli) chromosome. The origin point of
replication in F factor for transfer of DNA into the
recipient cell is called “ori T” (Fig. 18.2), while the
origin point at which replication of the F factor begins
as an autonomous unit (during cell division of the F1”
cells) is designated as “ori V” (Origin of vegetative
replication).

4. • The transfer operon (tra) is a large region (33 kb) of the F plasmid. It is
required for conjugation. The tra operon contains more than 25 genes
which are required for transfer of DNA. Most of these genes are
expressed coordinately as part of a 32 kb transcription unit. The
genes tra J and tra M are expressed separately. The F factor tra
operon has homology with the tra operon of R plasmid (F-like R
plasmids). It also contains IS elements that provide the sites for its
integration into the host chromosome.

5. • Mechanism of Transfer of F Factor:
• The F+ bacterial cells have surface appendages called
F-pili or sex-pili which are coded by the F plasmid. An
F+ cell has 23 pili. They are hair-like structures of
about 2-3 µm length and appear as extension from
the bacterial cell wall. A single subunit protein “pilin”,
coded by gene tra A polymerizes into “pilus”.

6. • The pilin is polymerized into a hollow cylinder of about 8 nm
diameter and 2 nm axial hole. The sex pilus is necessary for
contact between the donor and the recipient cell. These pili may
also serve as specific attachment sites for certain icosahedral
RNA bacteriophages (MS2, QB) and filamentous DNA
bacteriophages (M13, f9).
• When the tip of the F pilus contacts the surface of recipient (F–)
cell, mating is initiated. Mating cannot occur between two
F+ cells because the genes tra S and tra T code for “surface
exclusion proteins”. The Tra Y and/or Tra I makes a nick in one
strand of the F factor at the ori T point (origin transfer point).
Then this protein complex (Tra Y/Tra I) binds to DNA and causes
the unwinding of about 200 bp.

7. • The protein complex moves along DNA from 5′-end and
unwind DNA at the rate of 1200 bp per second. The 5′-end of
the cut end enters the recipient cell and is released when the
circle gets back to the origin, i.e., only the unit length of
plasmid DNA is transferred.
• If there is concomitant DNA replication the process becomes
a rolling circle type. After the transfer is completed, a
complementary strand is synthesized on the transferred DNA
strand into the recipient cell, and the plasmid is circularized.

8. • Integration of F Plasmid into the Bacterial Chromosome:
• Integration of the F factor into the bacterial chromosome occurs
at specific regions of DNA called insertion sequences (IS) (Fig.
18.2, 18.3). The F factor carries several IS regions, such as, IS 2, IS
3, V-δ. The E. coli chromosome carries 20 IS regions which are
scattered throughout the chromosome with both, clock-wise and
counter clock-wise orientations.
• The IS elements provide the loci for homologous pairing between
the F factor and the bacterial chromosome and a single crossover
event between them is sufficient for the integration of the F
factor into the bacterial chromosome (Fig. 18.3). The
chromosome carrying an F plasmid is called an Hfr chromosome.

9. • The Hfr factor has a stable attachment at a particular point in the host
chromosome. The Hfr factor can break the bacterial chromosome and
orient its transfer at different points to yield different Hfr strains. In
strain H, the attachment point of Hfr factor is near threonine (thr),
while in strain C, it is near lactose (lac) marker.
•

11. • F Mediated Transfer of Bacterial Chromosome:
• After effective contact and sex pili formation, the bacterial chromosome is
ready for transfer. The transfer starts after 8 minutes of effective contact
between the recipient and donor cells and it takes about 100 minutes for
the complete transfer of the E. coli chromosome.
• The entire bacterial chromosome can be mapped in 100 time units (1 unit =
1 minute), and the method is called conjugation mapping. Different Hfr
strains initiate the transfer of their chromosomes at different points
depending on the site of the F factor integration.
• For example, the strain Hfr H transfers the gene for threonine synthesis
(thr) first, strain Hfr J4 transfers the gene for thiamine synthesis (thi) first,
the strain Hfr C transfers the gene for lactose (lac) first, while the strain Hfr
G H transfers the gene xyl first. However, an F+ strain is not restricted to
transfer genes from only a single point but it transfers it randomly.

12. • In the Hfr chromosome, the integrated F-DNA is nicked at ori T to generate 5′ and
3′-ends. The nicked strand begins to separate from the 5′-end; a short sequence
of the F-DNA first enters the recipient cell from its 5′-end, followed by the
transfer of the donor bacterial chromosome.
• Thus one strand of the donor Hfr chromosome enters in the recipient cell from its
5′-end, while the 3′-end of this strand is utilized for rolling circle replication of the
Hfr chromosome in the donor cell (Fig. 18.4). Simultaneously, the DNA strand
being transferred into the F–recipient cell serves as a template for the synthesis of
its complementary strand.
• The process of transfer continues until the complete Hfr chromosome (including
the integrated F factor) is transferred into or the mating is interrupted due to
breaking of contacts between the conjugating bacteria. Recombination may occur
between the recipient chromosome and the donor DNA.
•

16. • Conjugation Mapping:
• Jacob and Wollmann developed the interrupted mating technique for
conjugation mapping. In this technique, the conjugating donor and
recipient cells are separated at different time intervals, using a waring
blender, and the genes transferred into the cell are determined by
detecting the appearance of appropriate recombinants in the population.
• It has been found that it takes 8 minutes for the start of the chromosome
transfer, and that genes thr and leu were the first to be transferred from
the Hfr H strain.
• The time taken for the transfer of thr and leu was 8½ minute (including the
initial 8 minutes). The gene azi entered the recipient cell after 9 minutes (8
+ 1 minutes) and was placed at one unit distance on the map. Thus in Hfr
H, the F factor is located after the methionine B locus (88 minutes) (Fig.
18.5). The functions of genetic markers shown in the Fig. 18.5, are given in
Table 18.2.

18. • F-Duction (Sex-Ductiori):
• The F factor can be separated from the Hfr DNA to produce an
independent F factor. In this process, the Hfr chromosome folds upon itself
to form a figure “8” so that the F segment becomes aligned as a ring due to
synapsis between the IS elements in the integration of the F factor into the
host chromosome.
• Now a crossing over within the paired IS elements leads to the formation
of an independent F factor and a normal bacterial chromosome. But
sometimes, synapsis of the IS element of the F factor may occur with a
bacterial IS element other than that previously used for the integration.
• In such situations, the separated F factors will contain a section of the
bacterial DNA; such F factors are called “F prime” (F) or “substituted F
factor” (Fig. 18.6). As a result, F’ factors can transmit bacterial genes from
the F+ cells to the F– cells. For example, F carrying the lac+gene will transmit
it to an F– lac– cell; as a result, the latter will be converted into a lac+ cell.
This process of gene transfer is called sexduction or F’ duction.

19. • Interspecific Transfer of Plasmids:
• Presence of similar genes on plasmids of distantly related bacteria
indicates that interspecific transfer of plasmids can occur in many
bacterial species. The sex factor or F plasmid of E. culi can be
transferred to such different species as Shigella, Salmonella and
Proteus. About 1/3 of freshly isolated E. coli strains have been found
to contain conjugative plasmids. Such plasmids are found in about 30
bacterial genera most of which are Gram negative.