Gene transfer Methods-Conjugation, Transformation, Transduction

1. Transfer of genetic information
in prokaryotes
Bijesh K
Research Scholar-Microbiology

2. Recombination
• is the process in which one or more nucleic acids
molecules are rearranged or combined to produce a new
nucleotide sequence.
Bacterial Recombination:
• Homologous or General recombination
• reciprocal exchange between a pair of homologous DNA sequences (two
similar or identical molecules of DNA)
• Illegitimate or nonhomologous recombination
• occurs in regions where no large-scale sequence similarity
• Site-specific recombination.
• The genetic material is not homologous with the chromosome
• important in the integration of virus genomes into bacterial chromosomes
• replicative recombination
• generates a new copy of a segment of DNA.
• transposable elements

4. Two Models For Homologous
Recombination
1.The Holliday Model
2.The Double-Strand Break Repair Model
Robin Holliday

6. Holliday junction
Heteroduplex

7. For finishing the recombination it requires resolution of the holiday junction- by
cutting
The Holiday junction is rotated to
give a square planar structure with
no crossing over

8. 2 alternative choice of cleavage
c
c
C
C
Splice recombinant/ Crossover product:
Cut occur in 2 intact DNA strands of (b)
Holiday Junction cleavage
Resolution
Patch recombinant/non crossover product
Cut occur in 2 cut DNA strands of (b)

12. The Double-Strand Break Repair Model
Ref: Molecular Biology of the cell- Bruce Alberts
3’overhang

13. RecBCD
3‘ 5‘
5‘ 3‘
RecA –ssbp-strand invasion
(find complementary seq. in
ds )
RuvA & B
Helicase &branch
migration
RuvC –nick
formation
The Double-Strand Break Repair Model

14. Crossover hot-spot instigator (Chi) sequences Ref: Molecular Biology of the Gene- Watson

16. • Plasmid?
• Classification?
• Episome?
• Curing?
• Transposons

17. Plasmid
• Small, circular pieces of DNA that are separated and replicated independently
from the bacterial chromosome.
• Contains only a few genes that are usually not needed for growth and
reproduction of the cell.
• But important in stressful situations
• F plasmid, facilitates conjugation
• Can give a bacterium new genes that may help forsurvival in changing
environment.
• An episome is a plasmid that can exist either with or without being integrated
into the host’s chromosome.
• plasmids can be eliminated from host cells in a process known as curing.
Transposons are segments of DNA that can move around to
different positions in the genome of a single cell---jumping genes

18. • Conjugative plasmids/ Fertility plasmid
Eg: F factor of E.coli---genes for sex pili and conjugation
• Metabolic Plasmids/ Degradative plasmids
genes for enzymes that catabolize unusual organic molecules
Eg: TOL plasmid : Pseudomonas species – Toluene degradation
• Col Plasmids
Plasmids carrying genes for toxins or bacteriocins production
Eg: ColE1of E.coli----Colicin E1 production
• R Plasmids
Plasmids carrying genes for resistance (R) factors
Eg: RP4 of Pseudomonas resistance to many antibiotics
• Virulence Plasmids
make their hosts more pathogenic- production of toxin
Eg: Ent (P307)-enterotoxigenic strains of E. coli cause traveler’s
diarrhea because of a plasmid that codes for an enterotoxin
Major Types of Plasmids

19. Plasmid replication
• Theta (θ )Mode–
• Rolling circle mode
• Strand displacement mode

20. • horizontal gene transfer/ lateral gene transfer:
• Vertical gene transfer

21. General Features of
Gene Transfer in Bacteria
• Horizontal transfer
– Donor to recipient
• Donor does not give an entire chromosome
– in the case of certain plasmid, the entire plasmid is
transferred)
• Bacterial genes are usually transferred to members
of the same species but occasionally transfer to
other species can also occur.
• Enhances genetic diversity
Eg: Confer resistance to antibiotic when one a antibiotic resistant
bacterium transfer the gene to another bacterial cell

22. Mechanism of Gene Transfer
Conjugation
Direct physical interaction between Donor and
recipient cell
Transformation
DNA is transferred as naked DNA/ direct intake of
DNA
Transduction
When virus infects a bacterium and transfer genetic
material

23. Conjugation

24. Conjugation
• Conjugation, the transfer of DNA between bacteria involving
direct contact/ or by a bridge-like connection between two
cells (conjugation tube)
• For the first time JOSHUA LEDERBERG & EDWARD TATUM in
1946 presented the evidence for bacterial conjugation
• Bernard Davis (1950)- need of physical contact
auxotrophic strains

25. Mating types in bacteria
• Donor
F factor (Fertility factor)-F+
Male
E.Coli F plasmid consists of 25 genes that mostly code for
production of sex pilli.
• Recipient
Lacks an F factor
F-female
• Transfer of plasmids to one cell to other cells is most frequently
mediated by conjugation.

26. F plasmid
The most common functional segments
constituting F factors are:
• OriT (Origin of Transfer): The sequence
which marks the starting point of
conjugative transfer.
• OriV (Origin of vegetative replication):
The sequence starting with which the
plasmid-DNA will be replicated in the
recipient cell.
• tra-region (transfer genes): Genes
coding the F-Pilus and DNA transfer
process.
• IS (Insertion Elements) composed of
one copy of IS2, two copies of IS3, and
one copy of IS1000: so-called "selfish
genes" (sequence fragments which can
integrate copies of themselves at
different locations)
which contains genes that allow the plasmids DNA to
be transferred between cells

27. Physiological States of F Factor
• Autonomous (F+)
– Characteristics of F+ x F- crosses
• F- becomes F+ while F+ remains F+
• Low transfer of donor chromosomal
genes
F+
• Integrated (Hfr)
– Characteristics of Hfr x F- crosses
• F- rarely becomes Hfr while
Hfr remains Hfr
• High transfer of certain donor
chromosomal genes F+ Hfr

28. • Autonomous with donor genes (F’)
– Characteristics of F’ x F- crosses
• F- becomes F’ while F’ remains F’
Hfr F’

29. Mechanism of F+ x F- Crosses
Pili shortens

30. • The first step in conjugation is the contact between donor
and recipient cells
• This is mediated by sex pili (or F pili) which are made only
by F+ strains
• These pili act as attachment sites for the F– bacteria
• Once contact is made, the pili shorten
• Donor and recipient cell are drawn closer together
• A conjugation bridge is formed between the two cells
• The successful contact stimulates the donor cells to begin
the transfer process

31. Mechanism of conjugation
2 components
1.transacting gene (Tra gene) and
2.Origin of transfer (Ori T) site
Dtr
DNA transfer and
replication
1. relaxases,
2. relaxosome
complex and
3. primase.
Mpf
(Mating pair
formation)
T
r
a
TraA, TraB, TraC, TraD,
TraE, TraF, TraG, TraH,
TraK, TraL, TraN, TraP,
TraQ, TraV, TraW, TraX
TraI, TraM, TraU, TraY
• Pilus
• Coupling
Proteins
• Channel
formation
• Dtr component prepare plasmid for transfer
• Mpf component holds the donor and recipient cell together, forms a channel through which
DNA is transferred and signal Dtr component to initiate transfer

32. Coupling protein
Mpf structure
Relaxase: TraI
• site specific endonuclease : create nick on OriT site
• transcribed along with the plasmid into the recipient
• recyclizes the plasmid after it has been transferred to the recipient cell
Relaxosome complex:
• it helps relaxase bind to the oriT site and initiates plasmid transfer,
• communicates with the coupling protein of Mpf component which signals relaxase
when to cut the plasmid at Ori t site,
• Helicase activity:helps to separate the plasmid DNA strands during displacement
and transfer of plasmid
Primase

33. Mpf (Mating pair formation) component
• Hold the donor & recipient cell together
• Formation of channel for DNA transfer
Pilus:
• holds donor and recipient cell together
Coupling proteins:
• It signals the relaxase which then initiates the process of DNA transfer
• determines which protein are to be transported to the recipient cell (
relaxase , helicase and primase)
• Channel: mediates the transfer of DNA from donor to recipient cell
Ori T site:
• It is the site where plasmid DNA transfer initiates in donor cell and the site
for recyclization in the recipient cell.
• It is the site which is specifically recognized by relaxase.

34. 1. The donor cell produces a pilus
2. Formation of mating pairs & the
channel is attached with various protein
deposition MPF & coupling
proteins
3. a signal from the coupling protein that
contact with a recipient has been made,
the relaxase protein initiates transfer
starting at the oriT site in the plasmid
(nick formation) & helicase then
separates the strands of the plasmid
DNA.

35. • The relaxase protein, which has
remained attached to the 5′ end of the
single-stranded DNA, is then
transported out of the donor cell
through the channel directly into the
recipient cell, dragging the attached
single stranded DNA along with it.
• The coupling protein pumps DNA
out of the donor cell
 Once in the recipient, the relaxase
protein helps recyclize the single-
stranded DNA
 Primase –primer strand for
complementary DNA synthesis
Mating pair separates & both the
donor and the recipient bacteria have
a double-stranded circular copy of the
plasmid
Ref….Molecular Genetics of Bacteria- Larry Snyder

36. Hfr x F- Crosses
What is an Hfr cell?
When F-plasmid (sex factor) integrated with
chromosomal DNA then such bacteria is known as high
frequency recombination (Hfr) bacteria.
Luca Cavalli-Sforza.
Recombination b/w IS2 elements in the plasmid and in the
chromosome, forming an Hfr cell.
1-IS2
2-IS3
1-IS1

38. Only a portion of genetic material is transferred (F- cell remain F- but –give new properties)

39. • On contact with a recipient cell, the DNA in the donor is nicked at a site in
the integrated plasmid
• one strand is displaced into the recipient cell (5’ )
• About 1.5 to 2 hours is required for the entire Hfr chromosome to pass
into the F – cell. Because most matings do not last that long, usually only a
portion of the Hfr chromosome is transmitted to the F – cell.
• Once inside the F – cell, the chromosomal material from the Hfr cell can
swap or recombine, with the homologous region of the recipient cell’s
chromosome.

40. Mapping the Genome… by interrupted mating
• A technique used to map bacterial genes by determining the
sequence in which donor genes enter recipient cells.
• The donor and recipient cells are physically linked through a sex
pili, which is synthesized by the donor cell. The pili is a very
fragile structure and break easily.
• The time it takes genes to enter the recipient cell is directly related
to their order along the bacterial chromosome
1. Mix donor and recipient cells. Hfr strs+ F-strr
2. Incubate to allow conjugation to get started
3. At time t, blend the culture in the kitchen blender. This disrupts
the cell pairs but does not break the individual cells.
4. Plate recipient cells (use streptomycin selection –why?).
5. Screen for recombinant markers.

41. sodium azide
bacteriophage T1

42. F’ x F- Crosses Prime Factors (F’)
SEXDUCTION or F-duction
Hfr F’
Formation of F’ by defective
excision

43. • both cells wind up with a copy of the episome & the recipiect cell become F'.
• it will be diploid for the segment of chromosomal DNA on the episome.
• Such a partially diploid bacterial cell is called a merozygote

44. • Just as in the F+/F- mating, both cells wind up with a copy of the
episome.
• The cell that was F- now has the F factor (along with the piece of
chromosomal DNA) and is therefore now F'.
• This cell, however, also has a complete chromosome, so it will be
diploid for the segment of chromosomal DNA on the episome.
• Such a partially diploid bacterial cell is called a merozygote.
• The chromosomal DNA on the episome can undergo
recombination at high frequency with its homologous sequence
on the chromosome.

45. After conjugation
• F+ mating with F- produces F+ and F+
• Hfr mating with F- produces Hfr and F-
• F' mating with F- produces F' and F‘
• A partially diploid bacterial cell is called a merozygote

46. Transformation
Definition: -is the direct uptake of exogenous DNA from its surroundings and
taken up through the cell membrane

47. • Factors affecting transformation
– DNA size and state
– Sensitive to nucleases
• Competence of the recipient --is the ability of a cell to alter its genetics by
taking up extracellular ("naked") DNA from its environment in the process
called transformation
• Competence factor: These competence-specific proteins include a
membrane-associated DNA-binding protein, a cell wall autolysin, and various
nucleases enables the cell to be transformed.
• Natural competence
• Induced /artificial competence
• At least 40 species of naturally competent and transformable bacteria have
been found
• Gram +ve : Bacillus subtilis, Streptococcus pneumonae etc.
• Gram –ve: H. infulenzae, N. gonorrahe, H. pylori, Cyanobacteria etc.
Discovered in 1928 by Frederick Griffith

48. R-strain
Non-capsulated-
avirulent
S-strain
capsulated-
virulent

49. In 1944, Oswald Avery Maclyn McCarty, and Colin MacLeod performed an
experiment to determine what Griffith's "transforming principle" was… DNA

50. • Any DNA that is not integrated into the chromosome will be degraded

51. Mechanism of bacterial transformation
4 important proteins/enzymes
• membrane-bound DNA binding protein.
• Nuclease
• ssb
• Rec A

52. Ref: Brock Biology of Microorganisms
Binding of ds DNA by a
membrane-bound DNA
binding protein.
Passage of one of the two
strands into the cell while
nuclease activity degrades the
other strand.
The single strand in the
cell is bound by specific
proteins (ssb).
recombination with
homologous regions of the
bacterial chromosome is
mediated by RecA protein
degraded

54. steps
(a) DNA binding:
– DNA comes first in the contact of cell surface of competent bacteria (First the
DNA binding is reversible and lasts for about 4-5 seconds. Thereafter, it
becomes irreversible permanently) bind with receptor sites & with the help of
certain DNA-binding protein
– 4-5 seconds. Thereafter, it becomes irreversible permanently. For about 2
minutes it remains in non-transforming state. Thereafter, before 5 minutes it is
converted into the transforming state
– Binding of dsDNA is depend on the microbe. For eg., in S. pneumoniae each
cell can bind only about ten molecules of double-stranded DNA of 10–15 kbp
each.
(b) Penetration & Uptake of ssDNA
– The DNA molecules that bind permanently enter the competent recipient cells
– The endonuclease-1 of the recipient cells which is associated with cell
membrane acts as DNA translocase by attacking and degrading one strand of
the dsDNA.
– So, only complementary single strand of DNA enters into the recipient cells
– The single stranded DNA is coated with SSB proteins (protect from nuclease
attack)

55. (c) Integration:
The bacterial protein like E. coli RecA protein probably facilitates the DNA
pairing during recombination-facilitates the local unwinding of dsDNA of the
recipient cell from the 5′ end.
The DNA is integrated into the genome of the recipient by recombination
The endonuclease cuts the unpaired free end of donor DNA or the recipient DNA.
This process is called trimming
The nick is sealed by DNA ligase
The protein machineries & the process of DNA
uptake is different in Gram positive & Gram
negative bacteria

56. The ComG proteins are proposed to form a structure that provides
access for DNA to the ComEA receptor through the peptidoglycan.
DNA would then be delivered to the ComEC–ComFA transport
complex. A DNA strand would be degraded by a nuclease, while its
complement is pulled into the cell by ComFA through an aqueous
pore formed by ComEC
Ref: Molecular Genetics of Bacteria.. Larry Snyder

57. The PilQ & PilE proteins are proposed to form a structure that
provides access for DNA to the ComE receptor through the
peptidoglycan. DNA would then be delivered to the ComA–PilT
transport complex. A DNA strand would be degraded by a nuclease,
while its complement is pulled into the cell by PilT through an
aqueous pore formed by ComA
Ref: Molecular Genetics of Bacteria.. Larry Snyder

58. in Salmonella typhimurium
Transduction
• Definition - Unlike transformation in which the naked DNA is
transferred in transduction DNA is carried by a bacteriophage.
or
• In transduction, DNA is transferred from cell to cell through the
agency of viruses

59. The filter in the U tube had
small enough pores to block
the movement of bacteria
between the two sides but
allowed the phage P22 to
pass
S. typhimurium

60. Phage Composition and Structure

61. Infection of Host Cells by Phages
• Adsorption
•Irreversible attachment
•Sheath Contraction
•Nucleic acid injection
•DNA uptake

62. Types
Virulent: (Lytic phage) capable of causing infection and eventually destruction
and death of the bacterial cell. These follow the lytic cycle. e.g. T4 host E.coli.
Temperate: does not cause destruptic infection instead phage DNA is incorporated
into bacterium DNA and is replicated with it and after some cycle become virulent
cause lysis. • e.g. lambda phage.
• Prophage– DNA that is incorporated in to the host
• Lysogeny - Is the process in which a virus incorporates its.
genetic material into the genome of its host.

63. Bacteriophage life cycle
Lytic Cycle
&
Lysogenic cycle

66. • The integrated phage DNA, called a prophage, is not active: its genes aren't expressed,
and it doesn't drive production of new phages
• Under the right conditions, the prophage can become active and come back out of the
bacterial chromosome and enter in to the lytic cycle

67. Transduction happens through either the lytic cycle or
the lysogenic cycle
Generalized Transduction:
• As a result of faulty head stuffing of the virus in lytic cycle
• As chromosomal DNA is broken, a piece can get packaged into a
virus.
• This virus can infect a new cell and transfer genes from the first
bacterium

68. 1. A lytic bacteriophage adsorbs to a susceptible
bacterium.
2. The bacteriophage genome enters the bacterium.
The genome directs the bacterium's metabolic
machinery to manufacture bacteriophage
components and enzymes
3. Occasionally, a bacteriophage head or capsid
assembles around a fragment of donor bacterium's
nucleoid instead of a phage genome by mistake
4. The bacteriophages are released.

69. 5. The bacteriophage carrying the donor
bacterium's DNA adsorbs to a recipient
bacterium.
6. The bacteriophage inserts the donor
bacterium's DNA it is carrying into the
recipient bacterium.
7. The donor bacterium's DNA is recombined
with recipient

70. Abortive transductants:
• About 70 to 90% of the transferred DNA is not integrated but often is able to
survive and express itself.
• are bacteria that contain this nonintegrated, transduced DNA and are partial
diploids
Exogenote
Endogenote

72. Specialized transduction:
• A DNA fragment is transferred from one bacterium to another by a temperate
bacteriophage that is now carrying donor bacterial DNA due to an error in
spontaneous induction during the lysogenic life cycle.
• The best-studied example of specialized transduction is the lambda phage. The
lambda genome inserts into the host chromosome at specific locations known
as attachment or att sites
• The att site for lambda is next to the gal and bio genes on the E. coli
chromosome;

73. 1. A temperate bacteriophage adsorbs to a
susceptible bacterium and injects it genome
2. The bacteriophage inserts its genome into the
bacterium's nucleoid to become a prophage.
3. Occasionally during spontaneous induction, a
small piece of the donor bacterium's DNA is
picked up as part of the phage's genome in place
of some of the phage DNA which remains in the
bacterium's nucleoid.
----usually y is defective and lacks some part of its
attachment site.
1
2
3

74. 3. .
4. As the bacteriophage replicates, the segment
of bacterial DNA replicates as part of the
phage's genome. Every phage now carries
that segment of bacterial DNA.
5. The bacteriophage adsorbs to a recipient
bacterium and injects its genome.
6. The bacteriophage genome carrying the
donor bacterial DNA inserts into the
recipient bacterium's nucleoid.
4
5
6

75. Ref: Prescott—P No. 310

76. lambda dgal because they carry the gal utilization genes & is a defective
low-frequency transduction lysates (LFT lysates): When the aberrant excision
initially occurs, it will yield less than one specialized transducing phage per
1,000,000 wild-type phage.

77. 1. stable transductants : lambda dgal infect a new gal- cell & becomes gal+
2. lambda dgal infect a new cell already infected with a normal phage-The normal
phage in this instance is termed the helper phage because it aids integration and
reproduction of the defective phage

78. • The transductants are unstable because the prophage can be excised by
certain stress like UV radiation
• Hence, induction of this double lysogen (dislysogen) produces high
frequency transduction (HFT) lysates containing about 50% λdgal and
50% phage λ.
• is very effective in transduction,

79. The mechanism of specialized transduction
Lambda genome structure
The lambda genome inserts into the
host chromosome at specific locations
known as attachment or att sites
(similar att sites are; present in
bacterial chromosome)
Integration with int (integrase)
Excision with , xis (excisionase)

80. More CI: lysogenic cycle
More Cro: lytic cycle
CII & CIII- helps the production
of CI

81. CL

82. Diff b/w lytic & lysogenic
Lytic
Any part of the b’al genome can be
transferred
Lysogenic
Only particular genome

83. Lytic cycle

84. Websites
• http://www.biologydiscussion.com/genetics/types-of-transduction-
generalized-and-specialized-genetics/65186
• Prescot –311
• https://www.quora.com/What-are-the-factors-that-lead-induction-from-the-
lysogenic-cycle-to-the-lytic-cycle-and-how-does-it-occur
• http://www.biologydiscussion.com/genetics/mechanism-of-transformation-
with-diagram-genetics/65239
• http://www.onlinebiologynotes.com/bacterial-transformation/
Books
Molecular Biology of the Gene, Sheela , Prescot,
Brooker, Larry Synder
REFERENCES