Transformation in Bacteria

Bacterial transformation is a process of

horizontal gene transfer by which
some bacteria take up foreign genetic material
(naked DNA) from the environment.
The condition for bacteria to
undergo transformation is its ability to take up
free, extracellular genetic material.
Such bacteria are termed as competent cells.
 Competent cells
• Competence refers to a temporary state of
being able to take up exogenous DNA from
the environment.
• It may be induced in laboratory
• Competence is specifically induced by DNA
damaging conditions.
• Natural genetic transformation appears to be
an adaptation for repair of DNA damage that
also generates genetic diversity.
 Naturally competent bacteria
Naturally competent bacteria carry sets of
genes that provide the protein machinery to
bring DNA across the cell membrane.
Due to the differences in structure of the cell
envelope between Gram-positive and Gram-
negative bacteria, there are some differences in
the mechanisms of DNA uptake in these cells,
however most of them share common features
that involve related proteins.
 Artificial competent Bacteria
Artificial competence in bacteria can be induced
in laboratory procedures that involve making
the cell passively permeable to DNA by
exposing it to conditions that includes
• incubation of cell in a solution
containing divalent cations often calcium
chloride
• under cold conditions,
• heat shock
 Electroporation
• Electroporation is method of promoting
competence. In this method the cells are
briefly shocked with an electric field of
10-20 kV/cm, which is thought to create holes
in the cell membrane through which the
plasmid DNA may enter. After the electric
shock, the holes are rapidly closed by the
cell's membrane-repair mechanisms.
Gram negative bacteria
In Gram-negative cells, due to the presence of an
extra membrane, the DNA requires the presence of
a channel formed by secretins on the outer
membrane.
Gram positive bacteria
In Gram positive bacteria electroporation is the
most commonly used transformation. It involves
the application of high voltage electric pulse of
short duration to induce the formation of transient
pores in cell walls and membranes
Natural Transformation
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. Transformation in Haemophilus
influenzae occurs most efficiently at the end of
exponential growth as bacterial growth
approaches stationary phase
Transformation, as an adaptation for DNA repair
• Logarithmically growing bacteria differ from
stationary phase bacteria with respect to the
number of genome copies present in the cell, and
this has implications for the capability to carry
out an important DNA repair process.
• The process of homologous recombinational
repair (HRR) is a key DNA repair process that is
especially effective for repairing double-strand
damages, such as double-strand breaks. This
process depends on a second homologous
chromosome in addition to the damaged
chromosome.
 History
• Transformation in bacteria was first
demonstrated in 1928 by the British
bacteriologist Frederick Griffith.
Griffith was interested in determining whether
injections of heat-killed bacteria could be used to
vaccinate mice against pneumonia
• In 1944 the "transforming principle" was
identified as being genetic by Oswald Avery, Colin
MacLeod, and Maclyn McCarty.
They isolated DNA from a virulent strain of S.
pneumoniae and made a harmless strain virulent.
• in 1970, Morton Mandel and Akiko Higa showed
that E. coli may be induced to take up DNA
from bacteriophage λ without the use of helper
phage after treatment with calcium chloride solution
• Two years later in 1972, Stanley Norman Cohen, Annie
Chang and Leslie Hsu showed that CaCl
treatment is also effective for transformation of
plasmid DNA.
• In the late 1980s transformation
using electroporation was developed , increasing the
efficiency of in-vitro transformation
• In 1982 transformation of animal and plant cells
was also investigated with the first transgenic
mouse being created by injecting a gene for a rat
growth hormone into a mouse embryo.
• In 1907 a bacterium that caused plant tumors,
Agrobacterium tumefaciens was discovered and
in the early 1970s the tumor-inducing agent was
found to be a DNA plasmid called the Ti plasmid.
 Mechanism of Transformation in
Bacteria
Bacterial transformation takes place in
number of steps
• Specially prepared bacteria are mixed with DNA e.
g., from a ligation.
• The bacteria are given a heat shock, which causes
some of them to take up a plasmid.
• Plasmids used in cloning contain an antibiotic
resistance gene. Thus, all of the bacteria are
placed on an antibiotic plate to select for ones
that took up a plasmid.
• Bacteria without a plasmid die. Each
bacterium with a plasmid gives rise to a
cluster of identical, plasmid-containing
bacteria called a colony.
• Several colonies are checked to identify one
with the right plasmid. e.g., by PCR or
restriction digest.
• A colony containing the right plasmid is grown
in bulk and used for plasmid or protein
production.
 Colonies need to be checked
• The bacteria that make colonies should all contain a
plasmid which provides antibiotic resistance. However,
it’s not necessarily the case that all of the plasmid-
containing colonies will have the same plasmid.
• When we cut and paste DNA, it's often possible for
side products to form, in addition to the plasmid we
intend to build. For instance, when we try to insert a
gene into a plasmid using a particular restriction
enzyme, we may get some cases where the plasmid
closes back up without taking in the gene, and other
cases where the gene goes in backwards.
• If the gene goes backwards, the wrong strand
of DNA would be transcribed and no protein
would be made.
• Because of these possibilities, it's important
to collect plasmid DNA from each colony and
check to see if it matches the plasmid we
were trying to build.
• Restriction digests, PCR, and DNA sequencing
are commonly used to analyze plasmid DNA
from bacterial colonies.