Bacterial Transformation

Bacterial transformation
Transformation is a process by which a recipient cell uptakes naked DNA from the surrounding
medium and recombines it into its own genome to acquire an altered genotype that is heritable.
DNA is derived from a donor bacterium and taken up by a recipient bacterium. If the
incoming DNA recombines with resident DNA in the cell, such as the chromosome, recombinant
types can form; the cell that has taken up the incoming DNA is referred to as a transformant.
The frequency of recombinant types for various genetic markers can be used for genetic analysis.
If the regions of two markers can be carried on the same piece of transforming DNA, the two
markers are said to be cotransformable. The higher the cotransformation frequency, the more
closely linked are the two markers on the DNA.
Discovery of bacterial transformation

Transformation was first demonstrated in Streptococcus pneumoniae by Fred Griffith in 1928.
The pathogenic bacteria are surrounded by a polysaccharide capsule that protects the bacteria
from the immune system of the infected animal. The capsule gives the bacterial colony a
glistening, smooth (S) appearance. The nonpathogenic bacteria lack the enzyme required to
synthesize the capsular polysaccharide and they form colonies that have rough (R) surface.
From the experiment mentioned above Fred Griffith concluded that the dead pathogenic bacteria
gave off a “transforming principle” that changed the live nonpathogenic rough-colony-forming
bacteria into the pathogenic smooth-colony form.
1|Page Dr. Subrata Kundu/ Bacterial

Transformation
Later in 1944 Avery, MacCarty and Macleod used the in vitro system to purify the “transforming
principle” and proved that it is DNA.
Natural transformation
Bacteria are the only organism known to transform naturally. Transformation does not occur
“naturally” in all species of bacteria. It occour naturally in those species that possess the
enzymatic machinery involved in active uptake and recombination process. The bacteria capable
of taking up transforming DNA are said to be competent.
Gram-positive bacteria: Bacillus subtilis; Streptococcus pneumoniae
Gram-negative bacteria: Haemophilus influenza; Neisseria gonorrhoeae; Helicobacter pylori
Competence: Competence is a physiological state that permits a cells to take up transforming

DNA and be genetically changed by it. It generally arises in the late log phase or just before
reaching the stationary phase.
Process of natural transformation Gram-positive bacteria

The com genes of gram positive bacteria are organized into several operons. The products of
several of these genes are involved in regulation of competence. The proteins encoded in the
comG operon form a pseudopilus that resembles type IV pili. Double stranded DNA interacts
directly with the pseudopilus. Double-stranded DNA at the cell surface is cleaved by the NucA
endonuclease into segments. The segments are then brought through the cell wall to the cell
membrane by retraction of the pseudopilus, which probably occurs by disassembly of the pilus
subunits. The comEA gene encodes protein that directly binds extracellular double stranded
DNA at the outer surface of the membrane. One strand of the DNA is then degraded, and the
other strand is transported through the membrane and into the cell through the ComEC channel,
using ComFA as an ATPdependent DNA translocase. Internalized ss DNA becomes immediately
resistant to exonucleases by binding of single stranded binding (SSB) protein.

Transformation
Uptake of DNA in Gram-negative bacteria
Efficient DNA uptake by these gram-negative bacteria requires the presence of a speciﬁc uptake
sequence termed as DNA uptake sequence (DUS) or uptake signal sequence (USS). In gram-
negative bacteria, the basic steps are: binding of double stranded DNA to the outer cell surface of
the bacterium, movement of the double-stranded DNA across the outer membrane and cell wall,
degradation of one of the DNA strands, and translocation of the remaining single strand of DNA
into the cytoplasm of the cell across the inner membrane.
Molecular mechanism of transformation

1. Aided by the RecA equivalent protein the incoming single-stranded fragment cause local
unwinding of the recipient chromosome presumably from the 5′ end
2. The displaced single strand is cut by unknown mechanism
3. The recipient DNA unwind at the end of the assimilated DNA. This process allow the base
pairing between recipient strand and the invading strand
4. Trimming nucleases remove the free end and the nick is sealed by the DNA ligase
5. The result is a heteroduplex region with a mismatched base pair
6. Repair of the mismatch base pair
Transformation
Artificial transformation
Chemical transformation: The DNA molecules pass only through any of several hundred
channels formed at the “adhesion zones”, where the outer and inner cell membranes are fused to
pores in the bacterial cell wall. The acidic phosphates of the DNA helix are negatively charged,
as are a proportion of the
phospholipids composing the
cell membranes and the
membrane pore. Thus
electrostatic repulsion between
anions may effectively block the
movement of DNA through the
adhesion zones. Treatment of
the cells at 0 ᴼC crystallizes the
fluid of the cell membrane,
stabilizing the distribution of
charged phosphates. The cations in
a transformation solution (Ca2+)
form complexes with exposed
phosphate groups, shielding the
negative charges. With this ionic
shield in place, DNA molecule
can then move through the
adhesion zone. Heat shock complement this process probably by creating a thermal imbalance on
either side of the membrane that physically helps to pump DNA through the adhesion zone.
Eclipse period: The period during which the transforming activity of potentially transformed
cells is temporarily lost is termed as eclipse period. The transforming activity is restored when
the transforming exogenous DNA is integrated into the recipient genome. The eclipse complex is
composed of single stranded DNA and specific DNA binding proteins that were synthesized
during competence development
Transformation
How would you determine if a type of bacterium you have isolated is naturally competent?
To determine whether a given bacterium is naturally competent, you would isolate an
auxotrophic mutant, such as a Met+ mutant, and mix it with DNA extracted from the wild type
bacterium. The mixture would then be plated on medium without methionine. The appearance of
colonies due to Met + recombinants would be evidence of transformation.
Certain gram-negative bacteria, including H. influenzae,

initially capture transforming DNA in membrane-
associated vesicles called transformasomes. These
vesicles contain the double-stranded DNA that has been
transported through the outer membrane and are likely to
represent the site at which the DNA is processed into
single-stranded form and transported through the inner
membrane.
Role of Natural Transformation

Nutrition: Organisms may take up DNA for use as a carbon and nitrogen source.
Repair: Cells may take up DNA from other cells to repair damage to their own DNA.
Recombination: The possibility that transformation allows recombination between individual
members of a species is also an attractive hypothesis but is difficult to prove.
Importance of natural transformation for forward and reverse genetics:

Transformation has been used in many bacteria to map genetic markers in chromosomes and to
reintroduce DNA into cells after the DNA has been manipulated in the test tube. This has made
naturally competent organisms, like B. subtilis, ideal model systems for molecular genetic
studies. This offers opportunities to construct many different types of mutations in genes,
including loss-, gain-, or change-of- function mutations. Such manipulations are more difficult in
bacteria that do not have efficient natural competence systems.

Transformation

Bacterial Transformation

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Bacterial transformation

Discovery of bacterial transformation

1|Page Dr. Subrata Kundu/ Bacterial

Competence: Competence is a physiological state that permits a cells to take up transforming

Process of natural transformation Gram-positive bacteria

2|Page Dr. Subrata Kundu/ Bacterial

Molecular mechanism of transformation

Certain gram-negative bacteria, including H. influenzae,

Role of Natural Transformation

Importance of natural transformation for forward and reverse genetics:

5|Page Dr. Subrata Kundu/ Bacterial

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