TRANSFORMATION IN BACTERIA, TYPES OF TRANSFORMATION IN GRAM POSITIVE & GRAM NEGATIVE BACTERIA,CHROMOSOME MAPPING USING TRANFORMATION

1. TRANSFORMATION
Submitted by : Tanishka
Course : MSc. Biotechnology
Year : Ist
Roll no. : 211206

2. TRANSFORMATION:
 Discovered by Fredrick Griffith in Streptococcus pneumoniae
(pneumococcus) 1928
 phenotypic characteristics of importance in Griffith’s demonstration
of transformation are
 the presence or absence of a polysaccharide capsule
 the type of capsule

3. Demonstrated :
Pneumoniae with
capsule
Pneumoniae without
capsule
Blood agar
medium in
Petri dish
Large ,smooth
colonies
[S]
Small ,rough colonies
[R]
Blood agar
medium in
petri dish
Grown
in
Grown
in
form
form

4. Characteristics :
Smooth colonies :
 virulent ( pathogenic ) in nature
 Causes pneumonia in mice and humans
Rough colonies :
 Non pathogenic in nature
Polysaccharide capsule is required for virulence because it protects the
bacterial cell from destruction by white blood cells.

5. Type of capsule on the specific molecular
composition of polysaccharides :
• Type I
• Type II
• Type III
Type II when injected into the blood stream of rabbit ,antibodies
production takes place ,agglutination occurs.
However, no agglutination was seen in Type I & Type III.
IDENTIFIED
IMMUNOLOGICALLY

6. Discovery by Griffith :
Image source : PRINCIPLES OF GENETICS BY SNUSTAD AND SIMMONS
CONCLUSION : Non encapsulated type R cells can
mutate back to encapsulated Type S cells

7. In such a mutation type II R should be converted into
type II S not type III S.
From here he concluded that component of dead type III
S have converted living type II R cells into type III S.

8. Type III S phenotype of transformed cells was passed on to progeny cells
It was due to of permanent inherited change in the genotype of the cells

9. Experiment by Richard Sia & Martin Daurson
• In 1931, performed the same experiment in vitro, showing
that the mice played no role in the transformation process.

10. GENETIC INFORMATION IS STORED IN DNA NOT PROTEIN
IMAGE SOURCE : SCIENCEDIRECT.COM
Performed by Maclyn McCarty, Colin MacLeod,
Oswald Avery in 1944
Image source : researchgate.net

11. Transformation Mechanism :
• Studied mainly in:
Bacillussubtilis
Streptococcus pneumoniae
Haemophilus influenzae
Neisseria gonorrhoeae

12. Competent cells : Cells that are expressing genes that encode proteins required
for the process are capable of taking up DNA.
Proteins that mediate the transformation process are known as Competent
proteins (Com ).
In bacteria , development of competence
 occurs during the late phase of their growth cycle (when cell density is high )
 Stops before the cell division
Image source :genetics a conceptual approach; Benjamin a. pierce

13. Natural competence in Bacillus subtilis :
Cells become competent –
• small peptides called competence pheromones are secreted by cells
& accumulate at high cell density
• High concentration of pheromones
Expression of genes encodes proteins that are required

15. Mechanism :
Step 1 :
Step 2 :
Step 3 :
Step 4 :
Com EA & Com G protein bind double stranded DNA
to the surface of competent cells
Bound DNA is pulled into the cell through the
channel composed of Com EC protein by Com FA
DNA translocase
One strand of DNA is degraded by
deoxyribonuclease
Single strand of transforming DNA invades the
chromosome of the recipient cell
Other strand is
protected from
degradation by a
coating of single
stranded DNA protein &
Recombinant protein A

16. Continued….
Step 5 :
Step 6 :
Step 7 :
Pairing with the complementary strand of
DNA & replacing the equivalent strand
Replaced recipient strand is then degraded
Formation of heteroduplex takes place
Will segregate into
homoduplexes when it
replicates

17. image source : Prescott, Harley & Klein’s microbiology

18. Natural competence in Neisseria gonorrhoeae
• is a gram negative bacteria.
• does not produce a competence factor to stimulate the development
of competence, and it takes up DNA from only closely related species.
• That contains a special short nucleotide pair sequence of 10 base pair.
• the machinery is quite large and complicated.
• A gram-negative equivalent of ComFA has not been identified yet.

19. Mechanism :
Step 1:
Step 2:
Step 3 :
Pil Q aids in the movement across the outer
membrane
pilin complex Pil E moves the DNA through the
periplasm and peptidoglycan
Com E is a DNA binding protein

20. Step 4 :
Step 5 :
Step 6 :
Step 7 :
Pairing with the complementary strand of
DNA & replacing the equivalent strand
Replaced recipient strand is then degraded
Formation of heteroduplex takes place
Will segregate into
homoduplexes when it
replicates
N is the nuclease that degrades one strand
before the DNA enters the cytoplasm
through the transmembrane channel
formed by Com A

21. image source : Prescott, Harley & Klein’s microbiology
DNA uptake machinery in B. subtilis Uptake machinery in N. gonorrhoeae

22. • Natural competence and gene transfer have facilitated many adaptations in prokaryotic and eukaryotic cells.
• example :
image source : researchgate.net
• eukaryotic red algae Galdieria sulphuraria;
gene transfer to this organism living in extreme environment came from prokaryotes via HGT.
The transferred genes led to a more versatile metabolism and the ability to detoxify mercury and arsenic.

23. Artificial competence:
• is the process by which a bacterial cell is artificially induced to take in
foreign DNA and incorporate it into its body.
There are two main methods for artificial transformation in bacteria;
 CaCl2 treatment followed by brief heat shock
 electroporation

24. Calcium chloride treatment :
• Methods for preparing the competent cells derived from the work of Mandel and Higa
who developed a simple treatment based on soaking the cells in cold CaCl2
• Rapidly growing cells are made competent more easily than cells in other Growth stages.
So it is necessary to bring cells into log phase before the procedure is begun. The cells in
rapid growth (log phase) are living, healthy, and actively metabolizing.
• divalent cations in cold condition changes or weaken the cell surface structure of the
cells making it more permeable to DNA.
• The heat-pulse creates a thermal imbalance on either side of the cell membrane, which
forces the DNA to enter the cells through either cell pores or the damaged cell wall.

25. Working :
• The surface of bacteria such as E. coli is negatively-charged due to phospholipids and
lipopolysaccharides on its cell surface, and the DNA is also negatively-charged
• naked DNA molecule is bound to the lipopolysaccharide(LPS) receptor molecules on the
competent cell surface.
• The divalent cations generate coordination complexes with the negatively charged DNA
molecules and LPS.
• DNA, being a larger molecule, cannot itself cross the cell membrane to enter into the
cytosol.
• The heat shock step strongly depolarizes the cell membrane of CaCl2-treated cells.
• Thus, the decrease in membrane potential lowers the negativity of the cell’s inside
potential which ultimately allows the movement of negatively charged DNA into the
cell’s interior.
• The subsequent cold shock again raises the membrane potential to its original value.

26. • This procedure is comparatively easy and simple, and can be
used in the genetic engineering of bacteria but in general
transformation efficiency is low.
• Efficiency of transformation can be increased by using high
concentration of DNA

27. Electroporation :
cells are made competent using an electrical pulse 10-20 kV/cm from
an electroporator to create temporary pores in the cell membrane of
either prokaryotic or eukaryotic cell
 While exogenous material can enter the cell due to increased
permeability, cellular material can also be lost during the process

28. Working :
• The phospholipid bilayer are dual chains of phospholipids
• phospholipids are composed of a hydrophilic head and a hydrophobic tail.
• The hydrophilic heads of the bilayer face outward: one row faces the
extracellular space, while the other row faces the intracellular fluid.
• The hydrophobic tails face each other inward, the internal portion of the
bilayer.
• When voltage from electroporation is applied, the electrical pulse
rearranges the orientation of the bilayer in a way that forms a gap through
which exogenous DNA enters.
• After the electric shock, the holes are rapidly closed by the cell’s
membrane-repair mechanisms

30. Differences :
Reversible electroporation (RE)
• involves a voltage of up to 1 kV.
• pores are temporary & resealed
• used to temporarily permeate
the cell membrane in order to
introduce foreign molecular
material, such as DNA, into a
cell.
Irreversible electroporation (IRE)
• involves up to 3 kV of DC
current.
• creates permanent pores that
ultimately leads to apoptosis.
• Researchers have been using
IRE for tumor treatment.
is not used for molecular
transformation and transfection

31. Other salts and chemicals used to make chemically
competent cells :
• CaCl2: Neutralizes the negative charges of the phospholipid bilayer and
DNA.
• DMSO: DMSO gathers at the hydrophilic heads of the lipid bilayer,
weakening the forces. As DMSO concentration increases, the thickness of
the bilayer decreases, increasing membrane permeability.
• MgCl2: Works the same way as CaCl2, but allows better DNA binding to the
cell.
• PEG: Shields the negative charges of the phospholipid bilayer and DNA.
• RbCl: Works the same way as CaCl2 and MgCl2. Some researchers prefer
RbCl when higher competency is required.

32. Chromosome mapping using transformation :
• Transformation can be used to measure
how closely two genes are linked on a
bacterial chromosome.
• If two donor genes are located close
together on the chromosome, there is a
good chance that they will be carried on
together in transformed cell, causing a
double transformation.
• Conversely, if genes are widely separated
on the chromosome, they will most likely
be carried on separate transforming
segments. Image source :
introduction to
genetic analysis;
griffith

33. Image source :genetics a conceptual approach; Benjamin a. pierce

34. In widely separated genes, the frequency of
double transformants will equal to the product of
the single-transformant frequencies.
if genes are linked, then the proportion of
double transformants will be greater than the
product of single-transformant frequencies.
Testing for close linkage from the product rule :
• Cant be used for all bacterial cell as all cells in a population of bacteria
are not competent .

35. References :
• https://international.neb.com/applications/cloning-and-synthetic-biology/transformation
• https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/7%3A_
Microbial_Genetics/7.11%3A_Genetic_Transfer_in_Prokaryotes/7.11B%3A_Bacterial_Transforma
tion
• https://www.mybiosource.com/learn/testing-procedures/cacl2-transformation-technique/
• https://www.goldbio.com/articles/article/Introduction-to-Competent-Cells
• PRINCIPLES OF GENETICS BY SNUSTAD AND SIMMONS
• Genetics a conceptual approach; Benjamin a. pierce
• Introduction to genetic analysis; Griffith
• Prescott, Harley & Klein’s microbiology
• www.researchgate.net
• www.Sciencedirect.com
• Concepts of genetics; Klug, Cummings, Spencer, Palladino

36. THANKYOU