Information about F plasmid and conjugation and types

1. The Role Model For Bacterial Conjugation
Present by – 1) Nikita Jedhe (218213)
2) Gunali Chaudhari (218238)

2. Discovery
 Esther M. Lederberg discovered Fertility factor
working with Joshua Lederberg.
 Lederberg's discovery of the fertility factor (F factor)
arise directly from her experiments to map the
location of lambda prophage on the E.
coli chromosome by crosses with other E. coli strains
with known genetic markers.
 When some of the crosses failed to give rise to
recombinants, she suspected that some of her E.
coli strains had lost a fertility factor.
 Later work by Hayes in 1952 showed that the F factor
is a bacterial DNA sequence harbouring genes that
allow a bacterium to donate DNA to a recipient
bacterium by direct contact in a process
called conjugation.
 The discovery of horizontal gene transfer between
bacteria can be attributed to the work of Lederberg
and Tatum (1946), who observed that different strains
of E. coli K-12 could be genetically and phenotypically
altered when mixed together.
 A series of experiments led to the conclusion that
direct contact between bacteria was required in order
for genetic material to be transferred between the
cells.
Experiment by Joshua Lederberg and Tatum

3. F Plasmid Map
 OriT (Origin of Transfer) - The sequence
which marks the starting point of
conjugative transfer.
 OriV (Origin of Vegetative Replication) -
The sequence starting with which the
plasmid-DNA will be replicated in the
recipient cell.
 tra-region (transfer genes) - Genes
coding the F-Pilus and DNA transfer
process.
 IS (Insertion Elements) - composed of
one copy of IS2, two copies of IS3, and
one copy of IS1000 - so-called "selfish
genes" (sequence fragments which can
integrate copies of themselves at different
locations).
 Some F plasmid genes and their Function -
traA: F-pilin - Major subunit of the F-pilus.
Genetic map of the F Plasmid

4. Conjugation
Types of bacterial conjugation F+ - F- Conjugation
In the conjugation between F+
cell and F-
cell, frequency of recombination is very
low but frequency of transfer of whole F
factor is very high.

5. Hfr – F-
Conjugation F’ – F-
Conjugation
In the conjugation between Hfr cell and F-
cell, frequency of recombination is very high
but frequency of transfer of whole F factor is
very low.
In the conjugation between F-prime (F’) cell
and F-
cell, frequency of recombination is
high as well as frequency of transfer of
whole F factor is also high.

6. Type IV Secretion System (T4SS) System
The Proteins necessary for conjugation assemble into two complexes : a
DNA-processing complex called the relaxosome and a complex
responsible for transfer the T4SS.
The relaxosome is an assembly of a protein called relaxase and a few
accessory proteins that bind a specific DNA sequence called oriT (origin
of transfer) to form a nucleoprotein complex .
The T4SS is a large (3 to 4 MDa) protein complex consisting of a transport
apparatus that spans the bacterial cell envelope , a pilus that extends from the
cell surface and mediates contact between cells , and a type IV coupling
protein (T4CP) that recruits the relaxosome to the secretion channel.
The relaxase has two activities transesterase/nicking activity and helicase activity.
The transesterase nicks the plasmid DNA strand for transfer (T-strand) at a
specific position within oriT. The relaxase and accessory proteins carry
translocation signals for recruitment of the transfer intermediate to the T4SS via
T4CP. Upon contact with a recipient cell, the T –strand is transported into the
recipient cell in ATPases – dependent manner.
During the translocation process, the T-strand is unwound from its
complementary strand by a second copy of the relaxase the helicase activity of
which motors the T-strand through the T4SS assisted by some of the T4SS
ATPases . In the recipient cell, the relaxase molecule that has passed through
the system may recircularize the T-strand and the complementary strand is
synthesized .

7. Structural Bases of F Plasmid Conjugation

8. FIN System
 The F-plasmid belongs to a class of conjugative
plasmids that control sexual functions of bacteria
with a fertility inhibition (Fin) system.
 In this system, a trans-acting factor FinO and
antisense RNAs, FinP, combine to repress the
expression of the activator gene TraJ.
 TraJ is a transcription factor that up regulates
the tra operon.
 The tra operon includes genes required for
conjugation and plasmid transfer. This means that
an F+ bacteria can always act as a donor cell.
 The finO gene of the original F plasmid (in E.
coli K12) is interrupted by an IS3 insertion, resulting
in constitutive tra operon expression.
 F+ cells also have the surface exclusion proteins TraS
and TraT on the bacterial surface. These proteins
prevent secondary mating events involving plasmids
belonging to the same incompatibility group.
 Thus, each F+ bacterium can host only a single
plasmid type of any given incompatibility group.
F- Plasmid of E.coli K12

9. Fosmid  Fosmids are vectors similar to cosmids but are
based on the bacterial F-plasmid used in genetic
engineering and gene therapy.
 Fosmids can hold DNA inserts of up to 40 kb in size.
 A fosmid library is prepared by extracting the
genomic DNA from the target organism and cloning
it into the fosmid vector.
 The ligation mix is then packaged into phage
particles and the DNA is transfected into the
bacterial host.
 Bacterial clones propagate the Fosmid library. The
low copy number of fosmid offers higher stability
than vectors like cosmids with relatively higher copy
numbers.
 The major advantage of Fosmids over other cosmid
systems lies in its capability of stably propagating
human DNA fragments.
 Fosmids have high structural stability and have
been found to maintain human DNA effectively even
after 100 generations of bacterial growth.
 Fosmid clones were used to help assess the
accuracy of the Public Human Genome Sequence.
Method of Preparation of Fosmid Library

10. Bacterial Artificial Chromosome (BAC)
• A bacterial artificial chromosome (BAC) is a DNA
construct, based on a functional
fertility plasmid used
for transforming and cloning in bacteria usually E.
Coli.
• F-plasmids play a crucial role because they contain
partition genes that promote the even distribution
of plasmids after bacterial cell division.
• The bacterial artificial chromosome's usual insert
size is 150–350 kbp .
• BACs are often used to sequence the genome of
organisms in genome projects for example
the Human Genome Project.
• A short piece of the organism's DNA is amplified
as an insert in BACs, and then sequenced.
• Used in genetic engineering and gene therapy.

11. References
 Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli Bo
Hu, Pratick Khara, and Peter J. Christie PNAS July 9, 2019 116 (28) 14222-14227; first
published June 25, 2019; https://doi.org/10.1073/pnas.1904428116
 https://www.onlinebiologynotes.com/high-frequency-recombination-hfr-cell-
conjugation-and-f-prime-f-cell/
 Williams, L. J., Tabbaa, D. G., Li, N., Berlin, A. M., Shea, T. P., Maccallum, I.,
Lawrence, M. S., Drier, Y., Getz, G., Young, S. K., Jaffe, D. B., Nusbaum, C., &
Gnirke, A. (2012). Paired-end sequencing of Fosmid libraries by
Illumina. Genome research, 22(11), 2241–2249.
https://doi.org/10.1101/gr.138925.112
 https://www.genome.gov/sites/default/files/tg/en/illustration/bac_bacterial_artificial_c
hromosome.jpg
 https://biologyreader.com/
 Principles of gene manipulation and genomics – 7th edition by Primrose and Twyman.