CONJUGATION
INTRODUCTION:
• Conjugation is the method of transfer of genetic material from one
bacterium to another placed in contact or by a bridge like connection.
• This method was proposed by Joshua Lederberg and Edward Tatum in
1946.
• During conjugation, one bacterium serves as the donor of the genetic
material, and the other serves as the recipient.
• It is a mechanism of horizontal gene transfer which occurs in both
transduction and conjugation.
• They also discovered that the F-factor can move between E.coli cells and
proposed the concept.
• This method happens with the help of a pilus.
• Some bacteria species, algae, fungi, protozoans follow this method.
CONJUGATION PROCESS
• Classical conjugation is regarded as bacterial equivalent of sexual
reproduction because it involves exchange of genetic material.
• However, it is not sexual reproduction, since no exchange of gametes
occurs and no generation of a new organism.
• They differ in some physiological or genetic characteristic and are
distinguished by biologists through the use of symbols such as + and - or
by letters of the alphabet if more than two forms occur.
• So, they follow pansexual mode of reproduction mainly in bacteria.
• The genetic information transferred is often beneficial to the recipient.
• Benefits may include antibiotic resistance, xenobiotic tolerance or the
ability to use new metabolites.
FERTILITY FACTOR:
• The fertility or F factor is a defining characteristic of F-plasmids.
• Esther M. Lederberg and Luigi L. Cavalli-Sforza discovered "F,"
subsequently publishing with Joshua Lederberg
• Due to its role in the genetic material transfer from one bacterium to
another, it is also known as the “sex factor.”
• The following functional segments make up F factors:
I. OriT (Origin of Transfer) is the sequence that acts as the origin of
conjugative transfer.
II. OriV (Origin of Vegetative Replication) is the sequence where the
plasmid-DNA will replicate in the recipient cell.
III. DNA transfer and the F-Pilus are coded for by the tra-region (transfer
genes).
IV. IS (Insertion Elements), also known as “selfish genes”, consist of one
copy of IS1000, a single copy of IS2, and two copies of IS3.
• The role of fertility factor is controlled by the cluster of 25 tra genes,
which is broadly classified into two types:
1. Mpf: It is an acronym of the term Mating pair formation. Mpf genes hold
the mating cells together and provide a passage for the DNA and protein
transfer through pilus and some channels, respectively.
2. Dtr: It is an acronym for the term DNA transfer and replication. Dtr is a
gene product engaged in the processing and transferring of plasmid DNA.
F PLASMID
PROCESS:
Bacterial conjugation or F+ x F- plasmid conjugation involves the following
steps:
1.
2.
3.
4.
•
Pilus formation
Physical contact between Donor and Recipient Cell
Transfer of F- plasmid
Synthesis of complementary strand
Pilus formation:
The donor cells(F+ cells) form a sex pilus and begin contact with Frecipient cells. The F-pili of the donor cell initiates the process of mating by
first binding with the outer membrane protein of the recipient cell.
• Physical contact between Donor and Recipient Cell:
The pilus forms a conjugation tube and enables direct contact between
the donor and the recipient cells.
• Transfer of F- plasmid:
The F-factor opens at the origin of replication. One strand is cut at the origin
of replication, and the 5’ end enters the recipient cell.
• Synthesis of complementary strand:
The donor and the recipient strand both contain a single strand of the Fplasmid. Thus, a complementary strand is synthesized in both the recipient and
the donor. The recipient cell now contains a copy of F plasmid and becomes a
donor cell
PROCESS OF CONJUGATION
Hfr CONJUAGTION:
• A high-frequency recombination cell (Hfr cell) is a bacterium with
a conjugative plasmid (for example, the F-factor) integrated into
its chromosomal DNA.
• The Hfr strain was first characterized by Luca Cavalli-Sforza. William
Hayes also isolated another Hfr strain independently.
• The integration of the plasmid into the cell's chromosome is
through homologous recombination.
• A conjugative plasmid capable of chromosome integration is also called
an episome (a segment of DNA that can exist as a plasmid or become
integrated into the chromosome).
• When conjugation occurs, Hfr cells are very efficient in delivering
chromosomal genes of the cell into recipient F− cells, which lack the
episome.
HIGH FREQUENCY RECOMBINATION CELL
PROCESS:
1. The insertion sequences on both the F factor plasmid and the
chromosome have similar sequences, allowing the F factor to insert itself
into the genome of the cell.
2. The Hfr cell forms sex pili a pilus and attaches to a recipient F- cell.
A nick in one strand of the Hfr cell’s chromosome is created.
3. DNA begins to be transferred from the Hfr cell to the recipient cell while
the second strand of its chromosome is being replicated.
4. The pilus detaches from the recipient cell and retracts. The Hfr cell
ideally wants to transfer its entire genome to the recipient cell.
5. However, due to its large size and inability to keep in contact with the
recipient cell, it is not able to do so.
6. The F- cell remains F- because the entire F factor sequence was not
received. Since no homologous recombination occurred, the DNA that
was transferred is degraded by enzymes.
7. In very rare cases, the F factor will be completely transferred and the Fcell will become an Hfr cell.
STEPS INVOLVED IN Hfr x F- PLASMID
COINTEGRATE FORMATION:
• Cointegrate plasmid formation is a special type of plasmid evolution, in
which one plasmid acquires traits for host adaptation by capturing a
second plasmid carrying potentially beneficial genes.
• Cointegrate plasmids are useful models for the study of plasmid evolution
if their evolutionary processes can be replicated under laboratory
conditions.
• The frequency of cointegrate formation was high when plasmid had
extensive homology to each other.
• It is due to the presence of insertion elements(ISSIS and ISSIT) in
transferred chromosome.
• Non conjugative plasmids and conjugative plasmids present in a bacterial
cell carry one or more copies of same transposable element.
• Because these copies are homologous DNA sequences, they serve as
substrates for recombination and cointegrate formation occurs.
• If one of the participating plasmids is nonconjugative and other is
conjugative plasmid, then cointegrate is a conjugative plasmid and can be
transferred.
• By this mechanism, nonconjugative plasmid can temporarily ride along
with conjugative plasmid and transferred from cell to cell
• It is seen in Escherichia coli, Bacillus subtilis, Salmonella typhimurium
and Agrobacterium species and in some bacteriophages.
PROCESS OF COINTEGRATE FORMATION
F’ PLASMID:
• Bacteria which contain F-factor and a part of chromosomal DNA
integrated in it is known as F-prime bacteria.
• They are the derivatives of Hfr cells.
• F’ cells are formed from Hfr cell during induction of F- factor from
chromosomal DNA in which F-factor carries a portion of
chromosomal DNA along with it.
• In the cross (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.
F’PLASMID
TRANSFER FROM F’ DONOR TO F- RECEPIENT:
1. F’– strain contains excised F-plasmid integrated with the chromosomal DNA
of the Hfr strain.
2. F– Strain only contains the bacterial nucleoid and functions as a recipient
cell.
3. This kind of conjugation is virtually identical, where the F’ plasmid enters
the F– strain without being incorporated into the recipient’s nucleoid.
4. Therefore, a recipient cell becomes an F’– strain and functions as a partially
diploid merozygote by carrying an F’– plasmid or possessing two sets of
genes.
TIME OF GENE MAPPING:
• Gene mapping can be done by interrupting the conjugation at multiple
times and taking account of which genes the recipient cell has and can be
also done by Hfr x F- mating.
• The transfer and integration of genes of the Hfr chromosome to the
recipient in bacteria is measured by interrupted mating and the map is
constructed on the basis of minutes required for the linear transfer of a
particular marker(s).
• In this technique donor Hfr and recipient F" strains are mixed and
allowed to conjugate for a short period of time.
• Then samples are removed at periodic intervals.
• It can be also obtained by deliberate interruption in DNA transfer by
violent agitation in a blender.
• The length of donor chromosome transmitted could then be determined
and mapped in terms of time units required for transfer.
• Transfer begins at replication origin of F in the Hfr chromosome and the
time of entry of a gene is at the time at which gene enters a recipient.
• Separation of a mating pair prevents further transfer and limit
recombinants for a particular time.
• It is known that 8 minutes are needed for conjugation to begin and then
chromosome is transferred slowly in terms of time units, one time unit
being equal to one minute.
• Number of recombinants starts increasing with increase in time.
GENE MAPPING IN E.coli
• The complete chromosome of E. coli is transferred in about 89 minutes
and therefore the bacterial chromosome is 89-time units in length.
• Genes which are 2–3-time units apart can be precisely mapped by this
method.
• Further mapping within the limits of 1-3 minutes is done by conventional
recombination methods.
REFERENCE:
1. Hartl, Genetics: analysis of genes and genome, James and Bartlette
learning, 2011, pg-313-315,319-320
2. Brian JB. Wood, W.H.N.Holzapfel, The Genera of Lactic Acid Bacteria,
Springer US,1992,Pg-194
3. https://thebiotechnotes.com/2019/06/24/gene-mapping-usingconjugation/#:~:text=The%20str%20r%20recipients%20cells,and%20gal
%20%2B%20after%2025%20minutes.
4. George P. Redei, Encyclopedia of Genetics, Springer Publications,
2008,pg-412
5. https://byjus.com/neet/an-overview-of-f-plasmid/
6. https://www.onlinebiologynotes.com/high-frequency-recombination-hfrcell-conjugation-and-f-prime-f-cell/
7. B C Peterson, H Hashimoto, and R H Rownd, Cointegrate formation
between homologous plasmids in Escherichia coli, Journal of
Bacteriology
8. https://byjus.com/biology/bacterial-genetics/