Genetic

Recombinati
on
Genetic Transfer
Movement of genetic material between unicellular and/or multicellular
organisms

Types of Genetic Transfer

• Vertical gene transfer Occurs during reproduction,
between generations of cells
• Horizontal gene transfer
Transfer of genes between cells of
the same generation
Horizontal Gene transfer
• Transfer of naked DNA from one bacterium to another
• Recipient cells acquire genes from DNA outside the cell
• DNA is taken up by cell and often recombines with genes
on bacterial chromosome
• Transformation may alter phenotype of recipient cells
• Works best when donor and receipt are closely related
• Discovered by Fredrick Griffith in 1928 while working
with Streptococcus pneumoniae
• Result in RECOMBINATION
• Frederick Griffith, an English bacteriologist searching for a vaccine
against bacterial pneumonia.
• R strain. When grown in a petri dish, the R bacteria formed colonies,
or clumps of related bacteria, that had well-defined edges and a
rough appearance (hence the abbreviation "R").
• The R bacteria were nonvirulent.
• S strain. S bacteria formed colonies that were rounded and smooth
(hence the abbreviation "S").
• The smooth appearance was due to a polysaccharide, or sugar-based,
coat produced by the bacteria.
• This coat protected the S bacteria from the mouse immune system,
making them virulent (capable of causing disease).
• Mice injected with live S bacteria developed pneumonia and died
 S R “S” Heat killed R + S heat killed
Blood sample
from the dead
mouse contained
living S bacteria!

R-strain bacteria must have taken up what he called a "transforming principle" from the
heat-killed S bacteria, which allowed them to "transform" into smooth-coated bacteria and
become virulent.
 Avery–MacLeod–McCarty experiment
Large cultures of heat-killed S cells
• washing away
• separating out
• enzymatically destroying the
other cellular components

Purified the transforming principle

• The purified substance gave a negative test for proteins, but
a strongly positive test for DNA.
• The elemental composition of the purified transforming
principle closely resembled DNA in its ratio of nitrogen and
phosphorous.
• Protein- and RNA-degrading enzymes had little effect on the
transforming principle, but enzymes able to degrade DNA
eliminated the transforming activity
Hershey- Chase
• The phages were simple particles composed of protein and DNA,
• The outer structures made of protein and the inner core consisting of DNA.
• One sample was produced in the presence of 35S, a radioactive isotope of
sulfur.
• Sulfur is found in many proteins and is absent from DNA, so only phage
proteins were radioactively labelled by this treatment.
• The other sample was produced in the presence of 32P a radioactive
isotope of phosphorous.
• Phosphorous is found in DNA and not in proteins, so only phage DNA (and
not phage proteins) was radioactively labelled by this treatment.
large amount of 32P appeared in the pellet, whereas almost all of
the 35S appeared in the supernatant.
Hershey and Chase concluded that DNA, not protein, was injected
into host cells and made up the genetic material of the phage.
Genetic Recombination
• Exchange of genes between two DNA molecules
• Forms new combination of genes on a chromosome
Along with mutations, contributes to genetic diversity
Homologous recombination
• Rec A proteins are essential
• Rec BCD – endonuclease and helicase
activity
• Single stranded binding protein binds with
nick

Detection of recombinant
• Colony Morphology
• Nutritional selection
• Antibiotics sensitivity
Mechanisms of Horizontal Transfer

• Genes are naturally transferred between bacteria using three

mechanisms
• Transformation
• Transduction
• Conjugation
 Bacterial Transformation
• High efficiency natural transformation occurs in
Acetobacter, Azotobacter, Bacillus, Streptococcus,
Haemophillus, Neisseria and Thermus
• Bacillus 20% cell develop competence for several
hours
• Streptococcus 100% cell acquire competence but
for short period
• E.coli and many gram negative are poor
transformants

Natural Competence
•Membrane associated DNA binding proteins
•A cell wall autolysin
•Nucleases
 Steps involved
1. DNA Uptake
2. Integration of DNA
• Competent cells binds 1000 times more DNA than
Non- competent cells
• Size of DNA 10-15 kb, 8kb transformed
• Reversible binding convert to irreversible
• Maximum transformation frequency is 20%,
generally 0.1-1%
• ds DNA binds with MSDBP
• Nuclease enzyme acts on ds DNA and degrade one strand
• ssDNA binds with competence specific proteins
prevents degradation of ssDNA
• ssDNA reached to chromosome and recA protein helps integration of DNA to
host DNA
 Transfection

• Bacterial transformation with bacterial virus

• If bacteriophage is lytic, transfection results in virus production
• Useful to study the mechanism of transformation and recombination
Conjugation
• F (fertility) factor is a conjugative plasmid transferred from cell to cell
• Replicate as circular plasmid
• F factor is an episome = genetic element that can insert into
chromosome
• The F plasmid is a low-copy-number plasmid ~100 kb in length, and is
present in 1–2 copies per cell
 Conjugation
Type of cells
F+ cells donor cells containing F
factor
F− cells recipient cells lacking F
factor
Hfr cells: (high-frequency strains)
– donor cells with F factor
integrated into the donor
bacterial chromosome
F prime (F′) cells: contain F
plasmid carrying some bacterial
genes
• It carries its own origin of replication, the oriV, and an origin of
transfer, or oriT and tra locous
• It replicates once per cell cycle and segregates to both daughter cells
in cell division
• It is not a reciprocal exchange of genetic information
• Both cells synthesize a complementary strand to produce a double
stranded circular plasmid and also reproduce pili; both cells are now
viable donor for the F-factor
• Benefits may include antibiotic resistance, xenobiotic tolerance or the
ability to use new metabolites
 F factor and Conjugation
• The transfer is mediated by a tube-like
structure called a pilus, formed between the
cells, through which the plasmid DNA passes.

• DNA transfer is unidirectional, by rolling

circle model. The lagging strand template
peels away… and is transferred to the
recipient.

• The leading strand template is replicated in

the donor while the lagging strand template
is replicated in the recipient
 Hfr and Conjugation
• F factor can integrate into chromosome via genetic
exchange
• Cells with the F plasmid integrated into the bacterial
chromosome are known as Hfr cells
• When an Hfr cell undergoes conjugation, the process
of transfer of the F factor is initiated in the same
manner as in an F+ cell
• However, because the F factor is part of the bacterial
chromosome, transfer from an Hfr cell also includes
DNA from the chromosome
• Hfr = high frequency of recombination
• Transfer begins within an integrated F
factor and proceeds in one direction

• A part of F is the first transferred,

chromosomal genes are transferred next,
and the remaining part of F is the last

• The conjugating cells usually break apart

long before the entire bacterial
chromosome is transferred, and the final
segment of F is almost never transferred

The recipient cell remains F-

F’ Cells = Derivatives of Hfr
cells where F plasmid has
disintegrated from host
chromosome and picks up Forma
tion o
some host genes next to F f F’ Ce
lls
plasmid integration sites

HFr recipient cells now have got three different types of DNA – its
own chromosomal DNA, some part of F plasmid along with some
part of donor’s chromosomal DNA
 Transduction

• Bacteriophage: bacterial infection

viruses
• Virulent phages: reproduce
through the lytic cycle, and always
kill the host cells.

• Temperate phages: inactive

prophage–phage DNA integrates
into bacterial chromosomes.
 Lytic Cycle
• At first bacteriophage
infects donor cell and
begins lytic cycle.

• When virus enter into

bacterial cell, virus hijack
host cell and synthesize
virus components such as
genome, enzymes, capsid,
head tail and tail fibers.

• Then viral enzyme

hydrolyses host cell DNA
into small fragments.
 Lysogenic Cycle
• Phages capable of only the lytic cycle are called virulent
• Lysogenic cycle: no progeny particles are produced, the
infected bacterium survives, and a phage DNA is
transmitted to each bacterial progeny cell when the cell
divides
• Those phages that are also capable of the lysogenic cycle
are called temperate
• Certain strains of Clostridium botulinum, which causes
botulism, express botulinum toxin from phage-tranduced
genes
• Shigella dysenteriae, which produces dysentery has
toxins that fall into two major groups, Stx1 and Stx2,
lambda phage
 Generalized Transduction
• if donor DNA from any region of chromosome have a chance to enter
into transducing bacteriophage then it is known as generalized
transduction.

• During a LYTIC infection, a transducing phage, such as P1 infecting E.

coli, accidentally packages a piece of the bacterial chromosome into a
virus particle instead of its own viral DNA.

• The phage carrying the bacterial DNA then delivers it to the recipient
cell when it tried to infect again.

• The injected bacterial DNA may then be inserted into recipient

chromosome by homologous recombination
Generalized Transduction cont…..
 Specialized transduction

• Bacteriophage transfer only a few restricted gene (DNA fragments)

from donor bacteria to recipient bacteria.
• Specialized transduction is carried only by temperate bacteriophage
which undergoes lysogenic cycle in donor cell.
• When such lysogenic cell is exposed to certain stimulus such as some
chemicals or UV lights, it causes induction of virus genome from host
cell genome and begins lytic cycle