Transformation and Conjugation

1. GENE TRANSFER MECHANISMS:
TRANSFORMATION
GRIFFITH’S EXPERIMENT
Dr. M. Sonia Angeline
Assistant Professor
KJC

2. GENE TRANSFER MECHANISMS
• Prokaryotes reproduce asexually by binary fission; they can also exchange genetic material by
transformation, transduction, and conjugation.
• Binary fission is a type of reproduction in which the chromosome is replicated and the
resultant prokaryote is an exact copy of the parental prokaryate, thus leaving no opportunity
for genetic diversity.
• Transformation is a type of prokaryotic reproduction in which a prokaryote can take up DNA
found within the environment that has originated from other prokaryotes.
• Transduction is a type of prokaryotic reproduction in which a prokaryote is infected by a virus
which injects short pieces of chromosomal DNA from one bacterium to another.
• Conjugation is a type of prokaryotic reproduction in which DNA is transferred between
prokaryotes by means of a pilus.

4. TRANSFORMATION
• Transformation is a process by which genetic information is transferred from one bacterium
to another.
• This phenomenon was first discovered by Frederick Griffith in 1928 in the Journal of
Hygiene.
• In 1944 reporting in the Journal of Experimental Medicine, Oswald Avery, Colin MacLeod,
and Maclyn McCarty showed that DNA was the molecule responsible for transformation.
• These two experiments were pivotal experiments showing that a material substance from
one organism can transform the inheritance pattern in another organism (Griffith, 1944.)
• Additionally, DNA is a molecule that stores and transmits the information responsible for
transforming bacteria (Avery, MacLeod, and McCarty, 1944.)
• These pivotal experiments demonstrated that the material substance DNA is responsible for
storing and transmitting inheritance.

5. In transformation, the prokaryote takes in DNA found in its environment that is shed by
other prokaryotes.
If a nonpathogenic bacterium takes up DNA for a toxin gene from a pathogen and
incorporates the new DNA into its own chromosome, it, too, may become pathogenic.
Transformation is a process by which foreign genetic material is taken up by a cell.
The process results in a stable genetic change within the transformed cell.
TRANSFORMATION

6. GRIFFITH EXPERIMENT & TRANSFORMING
PRINCIPLE
• Griffith experiment was a stepping stone for the discovery of genetic material.
• Frederick Griffith experiments were conducted with Streptococcus pneumoniae.
• During the experiment, Griffith cultured Streptococcus pneumoniae bacteria which
showed two patterns of growth.
• Griffith wasn't trying to identify the genetic material, but rather, trying to develop a
vaccine against pneumonia.
• In his experiments, Griffith used two related strains of bacteria, known as R and S.
• One culture plate consisted of smooth shiny colonies (S) while other consisted of rough
colonies (R).
• The difference was due to the presence of mucous coat in S strain bacteria, whereas
the R strain bacteria lacked them.

7. GRIFFITH EXPERIMENT
R strain:
• When grown in a petri dish, the R bacteria formed colonies, or clumps of related
bacteria, that had well-defined edges and a rough appearance (hence the abbreviation
"R").
• The R bacteria were nonvirulent, meaning that they did not cause sickness when
injected into a mouse.
S strain:
• S bacteria formed colonies that were rounded and smooth (hence the abbreviation
"S").
• The smooth appearance was due to a polysaccharide, or sugar-based, coat produced
by the bacteria.
• This coat protected the S bacteria from the mouse immune system, making them
virulent (capable of causing disease).
• Mice injected with live S bacteria developed pneumonia and died.

8. GRIFFITH EXPERIMENT
Griffith's experiment involved the use of two strains of pneumococcus – a deadly virulent
strain (S) or a non-virulent strain (R)
· When Griffith infected mice with the non-virulent bacteria (strain R), the mice survived
· When Griffith infected mice with the virulent bacteria (strain S), the mice died
· When Griffith infected mice with heat-killed virulent bacteria (strain S), the mice survived
as the bacteria had been killed
· When Griffith infected mice with a mix of heat-killed strain S and living strain R, the mice
were found to have died
From this Griffith’s concluded that the living R cells had somehow been transformed into
virulent S cells.
This indicated that there was some form of transferrable genetic material present within the
cells (i.e. DNA).

11. GRIFFITH EXPERIMENT
• Based on the observation, Griffith concluded that R strain bacteria had been
transformed by S strain bacteria.
• The R strain inherited some ‘transforming principle’ from the heat-killed S strain
bacteria which made them virulent.
• And he assumed this transforming principle as genetic material.

12. DNA AS GENETIC MATERIAL
 Griffith experiment was a turning point towards the discovery of hereditary material. However, it
failed to explain the biochemistry of genetic material.
 Hence, a group of scientists, Oswald Avery, Colin MacLeod and Maclyn McCarty continued the
Griffith experiment in search of biochemical nature of the hereditary material.
 Their discovery revised the concept of protein as genetic material to DNA as genetic material.
 Avery and his team extracted and purified proteins, DNA, RNA and other biomolecules from the
heat-killed S strain bacteria.
 They discovered that DNA is the genetic material and it is alone responsible for the
transformation of the R strain bacteria.
 They observed that protein-digesting enzymes (proteases) and RNA-digesting enzymes (RNases)
didn’t inhibit transformation but DNase did.
 Although it was not accepted by all, they concluded DNA as genetic material.

13. CONJUGATION
 Genetic recombination in which there is a transfer of DNA from a living donor bacterium to a
living recipient bacterium by cell-to-cell contact.
 In Gram-negative bacteria it typically involves a conjugation or sex pilus.
 Conjugation is encoded by plasmids or transposons.
 It involves a donor bacterium that contains a conjugative plasmid and a recipient cell that does
not.
 A conjugative plasmid is self-transmissible, in that it possesses all the necessary genes for that
plasmid to transmit itself to another bacterium by conjugation.

14. CONJUGATION
 Conjugation is the process by which one bacterium transfers genetic material to another through
direct contact.
 During conjugation, one bacterium serves as the donor of the genetic material, and the other
serves as the recipient.
 The donor bacterium carries a DNA sequence called the fertility factor, or F-factor.
 The F-factor allows the donor to produce a thin, tubelike structure called a pilus, which the donor
uses to contact the recipient.
 The pilus then draws the two bacteria together, at which time the donor bacterium transfers
genetic material to the recipient bacterium.

15. CONJUGATION
 Typically, the genetic material is in the form of a plasmid, or a small, circular piece of DNA.
 The genetic material transferred during conjugation often provides the recipient bacterium with
some sort of genetic advantage.
 For instance, in many cases, conjugation serves to transfer plasmids that carry antibiotic
resistance genes.
 Conjugation genes known as tra genes enable the bacterium to form a mating pair with another
organism, while oriT (origin of transfer) sequences determine where on the plasmid DNA transfer
is initiated by serving as the replication start site where DNA replication enzymes will nick the
DNA to initiate DNA replication and transfer.

17. CONJUGATION- F+ X F-
•The process of bacterial conjugation is based on the principle that the plasmid or any other
genetic material is transferred from the donor cell to the recipient cell through close physical
contact.
•Of all the conjugative plasmids, the F (fertility) plasmid of E. coli was the first discovered and is
one of the best-studied.
•The F plasmid is present in one or two copies per cell and is very large (about 100 kilobases).
•E. coli harboring the F plasmid are referred to as donor (F+ or male) cells and E. coli lacking
the F plasmid are referred to as recipient (F– or female) cells.
•Only donor cells are capable of transferring the F plasmid to recipient cells.

18. CONJUGATION
•For transfer of the F plasmid from donor to recipient, intimate contact between cells, resulting in
mating-pair formation, is required.
•DNA codes for the proteins that make up the sex pilus. It also contains a special site where DNA
transfer during conjugation begins
•The transfer of genetic material is then brought by membrane fusion of the two cells by the action of
different enzymes.
•Following the membrane fusion, the replication of donor DNA occurs and is transferred into the
recipient cell.
•If the F factor is transferred during conjugation, the receiving cell turns into an F^++start
superscript, plus, end superscript donor that can make its own pilus and transfer DNA to other cells.

20. HIGH FREQUENCY RECOMBINATION (HFR)
CELL CONJUGATION
•When F-plasmid (sex factor) integrated with chromosomal DNA then such bacteria is known as high
frequency recombination (Hfr) bacteria.
•In the cross (conjugation) between Hfr cell and F- cell, frequency of recombination is very high but
frequency of transfer of whole F-factor is very low.
•Hfr cell acts as donor while F- cell acts as recipient.

21. HFR CONJUGATION
•At first F-factor makes sex pilus that joins donor and recipient cell then F- factor opens as
replication origin then one strand is cut down.
•Now the 5’ end of this strand enters into recipient cell through conjugation tube.
•Since, replication origin lies somewhere in the middle of F- factor, portion of F-factor that lies at
5’ end enters first into recipient cell but the portion situated at 3’ end enters only when whole
chromosomal DNA enters into the recipient cell.
•To transfer whole chromosomal DNA, it takes 100 minutes in E. coli. In most of the cases, sex
pilus (conjugation tube) breaks before transfer of whole chromosomal DNA takes place. So,
frequency of transfer of whole F-factor is very low.
•After the cross between Hfr cell and F- cell, recipient cell remains recipient.
•In this conjugation, chromosomal DNA is always almost transfer from donor to recipient cell
together with portion of F- factor. So, frequency of recombination is high.

23. F –PRIME (F’) CELL
• Bacteria in which contains F-factor and a part of chromosomal DNA integrated in it is known as
F-prime bacteria.
• 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.
• If the F plasmid inaccurately excises from the chromosome after formation of an Hfr, it can take a
portion of the chromosome with it, which then becomes part of the plasmid itself. This form of
the F plasmid is called an F' (F prime).
• F’ Cells = Derivatives of Hfr cells where F plasmid has disintegrated from host chromosome and
picks up some host genes next to F plasmid integration sites