The document discusses various mechanisms of horizontal gene transfer in bacteria, including transformation, transduction, and conjugation. Transformation involves bacteria taking up free DNA from the environment. Transduction involves the transfer of bacterial DNA between cells using bacteriophages. There are two types: generalized transduction transfers random bacterial DNA, while specialized transduction only transfers DNA near the phage insertion site. Conjugation involves the transfer of plasmids between bacteria through direct cell contact via conjugation pili. These mechanisms allow bacteria to rapidly acquire new genes.

1. GENETRNSFER MECHNISMS
By
Dr. SIVA PRASAD.B.V.
Dept of Microbiology
Yogi vemana university, kadapa

3. Genetic Exchange
There are three different natural processes by which
bacteria can gain new genetic material (DNA).
 Transformation in which DNA is taken up from the
environment
 Transduction in which the transfer of DNA from one
bacteria to another is mediated by a bacteriophage.
 Conjugation in which a plasmid is transferred from
one bacteria to another.

4. Bacterial transformation is a process of horizontal
gene transfer by which some bacteria take up foreign
genetic material from the environment.
It was first reported in Streptococcus pneumoniae by
Griffith in 1928. DNA as the transforming principle was
demonstrated by Avery et al in 1944.
The process of gene transfer by transformation does
not require a living donor cell but only requires the presence
of persistent DNA in the environment.
The prerequisite for bacteria to undergo
transformation is its ability to take up free, extracellular
genetic material. Such bacteria are termed as competent
cells.
WHAT IS BACTERIAL TRANSFORMATION?

5. The factors that regulate natural
competence vary between various genera.
Once the transforming factor enters the
cytoplasm, it may be degraded by nucleases if
it is different from the bacterial DNA.
If the exogenous genetic material is
similar to bacterial DNA, it may integrate
into the chromosome. Sometimes the
exogenous genetic material may co-exist as a
plasmid with chromosomal DNA.

6. Demonstration of transformation
Transformation of avirulent Streptococcus pneumoniae to a virulent type.
Avery, MacLeod and McCarty 1944

8. Definitions
In molecular biology and genetics, transformation is
the genetic alteration of a cell resulting from the
direct uptake and incorporation of exogenous
genetic material from its surroundings through the
cell membrane(s).
For transformation to take place, the recipient
bacterium must be in a state of competence,
which might occur in nature as a time-limited
response to environmental conditions such as
starvation and cell density, and may also be induced
in a laboratory.

9. Transformation is one of three forms of horizontal
gene transfer that occur in nature among bacteria,
in which DNA encoding for a trait passes from one
bacterium to another and is integrated into the
recipient genome by homologous recombination;
the other two are transduction, carried out by means
of a bacteriophage, and conjugation, in which a
gene is passed through direct contact between
bacteria.

10. Transformation" may also be used to describe
the insertion of new genetic material into
nonbacterial cells, including animal and plant
cells; however, because "transformation" has
a special meaning in relation to animal cells,
indicating progression to a cancerous state,
the process is usually called "transfection"

11. In transformation, the genetic material passes
through the intervening medium, and uptake is
completely dependent on the recipient
bacterium.
Competence refers to a temporary state of being
able to take up exogenous DNA from the
environment; it may be induced in a laboratory.

12. What are the consequences of transformation
Existing genes can be extensively modified by exploiting
existing variation within a population.
It has been shown that this can be important for
proteins associated with host interactions in
pathogenic bacteria.
Target genes for antibiotics can be quickly modified and
antibiotic resistence can be established.

13. Natural transformation is a bacterial adaptation for DNA transfer that
depends on the expression of numerous bacterial genes whose products
appear to be responsible for this process. In general, transformation is a
complex, energy-requiring developmental process.
In order for a bacterium to bind, take up and recombine exogenous
DNA into its chromosome, it must become competent, that is, enter a
special physiological state.
Artificial transformation - chemical treatment. - physical treatment. -
enzymatic treatment.
Two types of transformation
Natural transformation

14. Artificial competence can be induced in laboratory
procedures that involve making the cell passively
permeable to DNA by exposing it to conditions that do
not normally occur in nature. Typically the cells are
incubated in a solution containing divalent cations under
cold conditions.
Yeast
Most species of yeast, including Saccharomyces
cerevisiae, may be transformed by exogenous DNA in the
environment. Several methods have been developed to
facilitate this transformation at high frequency in the
lab. Yeast cells may be treated with enzymes to degrade
their cell wall.
Methods and mechanisms of transformation
in laboratory
Bacterial

15. Not all bacteria are capable of taking up exogenous DNA from
their environment. The practical approach to acquire
competent cells is to make the bacterial cells artificially
competent using chemicals or electrical pulses.
Chemical induction of competence involves the following
steps:
chilling the cells in the presence of calcium phosphate to
make them permeable
incubation with DNA heat shock treatment at 42 °C for 60-
120 seconds that causes the DNA to enter the cells
Note: To endure the heat shock treatment, it is important the
cells used are in the log phase of growth Alternatively, the
bacterial cells are made permeable by subjecting them to
electrical pulses, a process known as electroporation.
COMPETENCE OF BACTERIA

18. Electroporation (using electric field) is now used to
transfer the foreign DNA into the fragile cells.The
electric pulses induce the formation of large pores in the cell
membrane. These pores give a passage through which the
foreign DNA can enter into the protolasts and thus, increase
the transformation frequency.
What is electroporation in genetic
engineering?

19. It is non-viral, non-toxic and can be used on all
cell types including mammalian, bacteria, algae,
plant and yeast. It can be used on cells in all forms,
in vitro or in vivo. In vitro is Latin for “within glass”
and includes suspension cell, tissue slice/whole
organ, and adherent cell.
What cell is used in electroporation?

20. Plants
A number of methods are available to transfer
DNA into plant cells.
Agrobacterium-mediated transformation is the
easiest and most simple plant transformation.
Plant tissue (often leaves) are cut into small pieces.
e.g. 10x10mm, and soaked for ten minutes in a fluid
containing suspended Agrobacterium. The bacteria
will attach to many of the plant cells exposed by
the cut.

21. The phenomenon of transformation has been
widely used in molecular biology. As they are easily
grown in large numbers, transformed bacteria may
be used as host cells for the following:
to make multiple copies of the DNA
in cloning procedures
to express large amounts of proteins and
enzymes
in the generation of cDNA libraries
in DNA linkage studies
WHAT ARE APPLICATIONS OF
TRANSFORMATION?

22. DNA used for transformation reaction
The concentration of DNA must be carefully quantified and the
same DNA must be used for all transformations.
Supercoiled DNA is most efficient for transformation compared to
linear or ssDNA that has the transformation efficiency of <1%.
During electroporation, the salts present in the preparation mix
may lower transformation efficiency. Limit the volume of plasmid
DNA to 1 µL per transformation.
Column-purified DNA is most suitable as it is devoid of
contaminants that interfere with transformation.
Ligation mixtures inhibit transformation as the ligases inhibit
electroporation of cells. The ligases must be heat-inactivated (65 °C
for 5 minutes) before the mixture is added to the cells.
WHAT FACTORS AFFECT
TRANSFORMATION EFFICIENCY?

23. Heat shock: Optimal heat shock set up is as follows:
42 °C for 45 seconds for PCR tubes or thin-walled tubes
37 °C for 60 seconds for microfuge tubes or thick-walled
tubes
General set up: 37 °C for 60 seconds
Time between transformation and plating: The
transformation efficiency is significantly decreased as the
time between the transformation reaction and the plating is
increased. This, however, also depends on the strain and the
plasmid used.
Freeze/thawing of cells: Activity of cells that are refrozen
and thawed is significantly reduced resulting in at least two-
fold decrease in transformation efficiency.

25. Transduction

26. Viral reproduction: the lytic cycle
Generalized schematic for
viral reproduction in a host
bacterium, through the lytic
cycle.
In the lytic cycle, the virus
(phage) multiplies in the host
cell and the progeny viruses
are released by lysis of cell.

27. Viral reproduction: the lysogenic cycle
Generalized schematic for
viral reproduction in a host
bacterium, through the
lysogenic cycle.
In the lysogenic cycle, viral
DNA is integrated into the
host genome and replicates
as the chromosome
replicates, producing
lysogenic progeny cells

28. Transduction
Transduction involves the exchange of DNA between bacteria using
bacterial viruses (bacteriophage) as an intermediate. There are two
types of transduction:
generalized transduction and specialized transduction, that differ in
their mechanism and in the DNA that gets transferred.
When a phage infects a bacterial cell, it injects its DNA into the cell.
The viral DNA is replicated numerous times, and viral genes are
expressed, producing the proteins that make up the viral capsid (or
protein coat) and nucleases that digest the host genome into
fragments. The newly replicated viral DNA molecules are packaged
into viral capsids, and the bacterial cell is lysed (burst, and therefore
killed), releasing hundreds of viral progeny, which then go on to infect
other cells.

29. Generalized Transduction
Sometimes, during bacteriophage replication, a mistake is
made, and a fragment of the host DNA gets packaged into
a viral capsid. The resulting phage would be able to infect
another cell, but it would not have any viral genes, so it
would not be able to replicate. The cell infected by this
phage will survive, and would have an extra piece of
bacterial DNA present, which could undergo
recombination with the host chromosome, and perhaps
cause a gene conversion event. Because it is a random
fragment that gets packaged into the viral capsid, any
segment of the bacterial DNA can be transferred this way
(hence the name 'generalized').

30. Generalized transduction: Lytic phage

31. Specialized Transduction
Specialized transduction occurs only with certain
types of bacteriophage, such as phage lambda.
Lambda has the ability to establish what is called
a lysogenic infection in a bacterial cell. In a
lysogenic infection, the viral DNA becomes
incorporated into the host chromosome.

32. In a lysogenic infection by lambda, the DNA integrates into a
very specific spot in the host chromosome. The integrated viral DNA can
remain integrated for long periods of time, without disturbing the cell.
Under the appropriate conditions the viral DNA will excise itself from
the chromosome, and enter the lytic phase. The cell gets lysed, and new
bacteriophage particles are released to infect other cells.
Sometimes the excision of lambda is sloppy, and some bacteria
DNA is excised along with it. When the resulting virus infects another
cell, it will pass that bacterial DNA into the cell, along with its own
DNA. Because the viral DNA integrates into a specific location, when it
excises, the bacterial DNA removed with it will be the same in all cases.
Therefore, the DNA transferred to the second cell will be the same
segment of the bacterial chromosome. This is why this process is
called 'specialized' transduction.

33. Specialized transduction: Lysogenic phage

34. CONSEQUENCES OF TRANSDUCTION
 Specialized transduction can only transfer genes that
flank the specific insertion site and as such do not
contribute many new genes to the bacteria.
 Generalized transduction can be instrumental in the
transfer of 50-100 new genes and make dramatic
changes to the properties of the bacteria.
Transduction plays an important roll in the transfer
of, antibiotic resistence and pathogenicity factors.
This can be a deadly combination.

36. It is a process in which plasmids are transferred
by themselves alone or along with other DNA
element from one cell to another cell through
conjugation tube.
The cell which transfer plasmid is called donor and
the cell which receive the plasmid is
called recipient.
The cell which has received the plasmid from the
donor cell is called trans-conjugant.
The phenomenon of conjugation in bacteria was
discovered by laderburg and Tatum in 1946.
CONJUGATION

37. Types of Plasmids
1. Self-transmissible (F-plasmid) plasmid:
These plasmid encodes all the functions necessary for their
transfer as well as the transfer of other DNA element and
mobilizable plasmid into recipient cell.
These plasmid contains both Tra gene and Ori T sites
These plasmid are known as F-factor or F-plasmid or conjugative
plasmid
Present in Pseudomonas, E. coli, Bacillus, streptococcus,
Staphylococcus, Streptomyces etc

38. 2. Mobilizable plasmid:
These plasmid encodes only function for its transfer into
recipient cell. mobilizer plasmid is not transfer by itself. It
requires the help of self-transmissible plasmid for its
transfer.

39. Steps of bacterial conjugation
Step I: Pilus formation
Donor cell (F+ cell) produces the sex pilus, which is a structure that projects out
of the cell and begins contact with an F– (recipient) cell.
Step II: physical contact between donor cell and recipient cell
The pilus enables direct contact between the donor and the recipient cells
forming conjugation tube
Step III: transfer of F- plasmid
F-factor opens at replication origin (Ori T site).
one strand of F-factor is cut down at origin and then 5’end of this strand enters
into recipient cell.
Step 4: complementary strand synthesis
In the last step, the donor cell and the recipient cell, both containing single-
stranded DNA of F-plasmid. A complementary strand is then synthesized in
both donor and recipient cell, Now the recipient cell also contain a copy of F-
plasmid and become a donor cell.

41. Mechanism of conjugation
Conjugation is brought about by 2 genes in self transmissible
plasmid, namely transacting gene (Tra gene) and Origin of
transfer (Ori T) site.
I. Tra gene:
Tra gene consists of 2 components- Dtr and mpf
Dtr (DNA transfer and replication) component:
Dtr component prepare plasmid for transfer.
It includes components such as= relaxases, relaxosome complex
and primase.
Relaxase:
Relaxase is a site specific endonuclease which acts on plasmid at
its OriT site.
Relaxase also recyclizes the plasmid after it has been
transferred to the recipient cell.
Relaxase is transcribed along with the plasmid into the recipient

42. Relaxosome complex
 It consists of group of proteins clustered around the Ori T site
 Relaxosome carries three basic functions-
 It helps relaxase bind to the oriT site and initiates plasmid transfer,
 relaxosome communicates with the coupling protein of Mpf
component which signals relaxase when to cut the plasmid at Ori t site,
 Helicase is a component of relaxosome which helps to separate the
plasmid DNA strands during displacement and transfer of plasmid.
Primase:
 Primase has no role in replication replication of donor plasmid in donor
cell
 The free 3-OH end created at the nick site acts as a primer in donor cell.
 Primase is trnasfered to the recipient cell and synthesizes a primer to
complete the replication of another strand of plasmid DNA in recipient
cell.

43. Mpf (Mating pair formation) component:
Mpf component holds the donor and recipient cell together, forms a channel
through which DNA is transferred and signal Dtr component to initiate
transfer. It has 3 components- pilus, channel and coupling protein
Pilus:
Pilus holds donor and recipient cell together. It is 10nm in diameter tubular
structure with a central channel projecting out of the cell surface. Pili may
be structurally long, thin and flexiable and it is encoded by F-plasmid in
those cell
Incompatibility F-plasmid (Inc F) is a long and rigid pili encoded by
pKM101 (Inc N)
Short, thick and rigid plasmid is encoded by RP4 (Inc P)
Long, thin and flexible pili mediates conjugation in cell in liquid medium
Short, thick and rigid pili mediates conjugation in cell fixed to solid
support (Agar medium)
Inc I plasmid (col 1BP9) encodes both long, thin, flexible pili and short,
thick and rigid pilli, therefore it can mediate conjugation in both liquid and
solid media

44. Channel:
Channel are also encoded by Tra gene
Channel mediates the transfer of DNA from donor to recipient
cell.
Coupling proteins:
Coupling protein is associated with channel
It signals the relaxase which then initiates the process of DNA
transfer. Coupling protein determines which protein are to be
transported to the recipient cell ( relaxase and primase)
Ori T site:
It is the site where plasmid DNA transfer initiates in donor
cell and the site for recyclization in the recipient cell.
It is the site which is specifically recognized by relaxase.
Any plasmid that possesses Ori T site can be transferred with
the help of self-transmissible plasmid
Ori T site is a cis-acting site
A known Ori T site of F-plasmid has around 300 bp and
contains inverted repeats and AT rich sites.

46. Consequences of conjugation
A bacteria cell can get many new genes and in turn new
genetic properties when it gets a new plasmid.
In some cases these can be incorporated into the
genome by recombination and they thus become part
of the genome (plasmids can be lost).
Plasmids play an important roll in the transfer of
antibiotic resistence between bacteria. A deadly
combination if they are pathogenic.

47. It is the process of deliberately introducing naked or
purified nucleic acids into eukaryotic cells.
 It may also refer to other methods and cell types,
although other terms are often preferred:
"transformation" is typically used to describe non-
viral DNA transfer in bacteria and non-
animal eukaryotic cells, including plant cells.
 In animal cells, transfection is the preferred term as
transformation is also used to refer to progression to a
cancerous state (carcinogenesis) in these cells.
Transduction is often used to describe virus-mediated
gene transfer into eukaryotic cells.
Transfection

49. The methods are divided into 3 categories:
1. Chemical methods
Calcium Phosphate - Lipids - Cationic polymer
2. Physical methods
- Electroporation
- Microinjection
- Laserfection
- Sonoporation - Biolistic particle delivery Methods of
Transfection
3. Biological method - Virus-based

51. Advantages:
1. Deliver nucleic acids to cells in a culture dish with high
efficiency.
2. Easy to use, minimal steps required; adaptable to high-
throughput systems.
3. Using a highly active lipid will reduce the cost of lipid
and nucleic acid, and achieve effective results.
Disadvantage:
Not applicable to all cell types 1. Lipid-Mediated
Gene Delivery ● Also referred as lipofection or liposome-
based gene transfection. ● Mode: Uses lipids to cause a
cell to absorb exogenous DNA.

52. The main difference between transfection and
transformation.
Is that the transfection refers to the introduction of
foreign DNA into mammalian cells while the
transformation refers to the introduction of foreign DNA
into bacterial, yeast or plant cells.

53. Transformation Transfection
Applicable to bacteria Applicable to eukaryotic cells*
Exogenous genetic material is taken
up by competent bacteria
Exogenous genetic material is
introduced into the eukaryotic cells
Bacteria can be made competent
either chemically or by electroporation
Introduction of exogenous genetic
material may be liposome-mediated,
by electroporation or by using viral
vector
The exogenous genetic material may
integrate into the bacterial genome or
exist as a plasmid
The exogenous genetic material is
either integrated into the genome or is
degraded
Transformation enables the expression
of multiple copies of DNA resulting in
large amounts of protein or enzyme
that are not normally expressed by
bacteria
Genetic material of transformed
bacteria may be used to transfect
eukaryotic cells for DNA or protein
expression studies
COMPARISON OF TRANSFORMATION AND TRANSFECTION