1) The document discusses the process of natural transformation in bacteria, where DNA is transferred from one bacterial cell to another without direct contact. 2) It describes how competent bacterial cells can take up extracellular DNA released from lysed donor cells, and how the DNA can recombine into the recipient cell's genome. 3) The document compares natural transformation between gram-positive bacteria like Streptococcus pneumoniae and gram-negative bacteria like Haemophilus influenzae, noting differences in how DNA is taken up and the role of specific DNA sequences.

1. Dna uptake, Entry, and
Establishment in recepient cell
Shreya M Modi.
MSc Sem-2
Roll no.-11

2. Content

3. Introduction
• It is a process of genetic transfer that does not require
physical contact between the donor and recepient
cells.
• In the process of transformation the donor cell
lysis, releasing its DNA.

4. DNA UpTake
• The process of taking DNA into the recepient
cell through the process of transformation from
the donor’s cell.
• Donor cell – The cell whose gene material is
transformed to the donor cell.
• Recepient Cell- the cell who receives the genetic
material.

5. Competent cell
• When bacteria lyse, they release considerable amounts of DNA
into the surrounding environment. These fragments may be
relatively
large and contain several genes.
• If a fragment contacts a competent cell, one able to take up DNA
and be transformed, it can be bound to the cell and taken inside.
• The transformationfrequency of very competent cells is around
103 for most genera when an excess of DNA is used.
• That is, about one cell in every thousand will take up and integrate
the gene. Competency is a complex phenomenon and is dependent
on several conditions.
• Bacteria need to be in a certain stage of growth; for example, S.
pneumoniae becomes competent during the exponential phase
when the population reaches about 107 to 108 cells per ml. When a
population becomes competent, bacteria such as pneumococci
secrete a small protein called the competence factor that stimulates
the production

6. Natural transformation in gram
negative bacteria
• Natural transformation has been observed in some gram negative
bacteria. Transformation in Haemophilus influenzae, the first gram
negative bacterium in which natural competence was found, is different at
least two important respects from that in B. subtilis.
• DNA uptake is associated with the formation of small membraneous
structures, called transformasomes, which protrude outside the cell. The
transforming DNA is taken into these vesicles where it is then internalized
into the cell. One of the two strands is degraded while the remaining
strand may recombine with the host chromosome.
• Unlike gram positive bacteria, DNA uptake in gram negative bacteria
appears to require or involve the recognition of specific sequences. The
sequences or some bacteria are as follows:

7. Transformation in Haemophilus
influenzae
• Transformation in Haemophilus influenzae, a gram-
negative bacterium, differs from that in S. pneumoniae in
several respects.
• Haemophilus does not produce a competence factor to
stimulate the development of competence, and it takes up
DNA from only closely related species (S. pneumoniae is
less particular about the source of its DNA). Double-
stranded DNA, complexed with proteins, is taken in by
membrane vesicles. The specificity of
• Haemophilus transformation is due to a special 11 base
pair sequence (5′AAGTGCGGTCA3′) that is repeated over
1,400 times in H. influenzae DNA. DNA must have this
sequence to be bound by a competent cell.

9. • The mechanism of transformation has been intensively studied in S.
pneumoniae.
• A competent cell binds a double-stranded DNA fragment if the
fragment is moderately large; the process is random, and donor
fragments compete with each other. The DNA then is cleaved by
endonucleases to doublestranded fragments about 5 to 15
kilobases in size.
• DNA uptake requires energy expenditure.
• One strand is hydrolyzed by an envelope-associated exonuclease
during uptake; the other strand associates with small proteins and
moves through the plasma membrane.
• The single-stranded fragment can then align with a homologous
region of the genome and be integrated.

11. Artificial transformation
• Artificial transformation has been demonstrated in a
number of bacterial species, most notably in E. coli, where
it is used routinely for cloning DNA. However, even in
E. coli, the process or mechanism is not well understood.
• E. coli cells can be made competent for transformation
simply by treating them with calcium chloride. But, even
this simple treatment is a bit of an art and variables such as
the temperature of growth and the density of the culture
seem to be important in determining competence.

12. Key Steps for Transformation
Protein of interest
Protein for
antibiotic resistance
Plasmid DNA enters the bacterial cell and the genes are expressed.
• Bacterial cell suspension is placed in CaCl2 solution
• Cells must be in log phase of growth.
• Cells are kept on ice until heat shock treatment
• Heat shock at 42 ˚C for one minute
• Recover period in LB broth
• Cells are spread on appropriate selection plates

13. TRANSFORMATION BY ELECTROPORATION

14. CHEMICAL TRANSFORMATION WITH
CALCIUM CHLORIDE

15. References
• Microbiology, 5th Edition
By Lansing M. Prescott