Bacterial Conjugation

GENETIC MECHANISM OF BACTERIAL CONJUGATION

2. It is the process by which genetic elements contained in two separate
genomes are brought together in one unit.
Mechanism enable the organism to carry out some new function and
result in adaptation to changing environments.
Usually involves much larger changes:
Entire genes, sets of genes, or even whole chromosomes,
are transferred between organisms.
Genetic recombination

3. 1.Transformation: Which involves donor DNA freein the
environment.
2. Transduction: In which the donor DNA transfer is
mediated by a virus (Bacteriophages).
3. Conjugation: In which the transfer involves cell-to-cell
contact and a conjugative plasmid in the donor cell
Processes of genetic recombination in
prokaryotes

5. Bacterial Conjugation

6. History of Bacterial Conjugation
Discovered by Lederberg and Tatum (1946)
Conclusion: One
cell should transfer
some genetic
material to other

7. Lederberg and Tatum: More Work
Transfer is unidirectional
Some strains are always donors, some always recipients in
an exchange
Donor strains designated F
+
(fertility) and that of the recipient
strains are designated as F
-

8. Bernard Davis
Demonstrated using a
U-tube culture that,
contact between donor
and recipient cells was
necessary for the
transfer of genetic
material.

9. Bacterial Conjugation
(L. conjugatus: to unite or yoke together.)
“ Bacterial Conjugation is genetic recombination in which
there is a transfer of DNA from a living donor bacterium to a
recipient bacterium by mechanism involving cell-to-cell
contact.”
Process encoded by plasmid
And plasmid encode for the pili which are required for the
cell-to-cell contact.

10. Plasmid
Non transmissible plasmid
Lacks genes necessary for effective contact (tra) and
DNA transfer (mob)
Conjugative plasmid
Carry genes necessary for effective contact
Contain onlytragene

11. Mobilizable plasmid(Mob plasmid)
Contain mobgene
Can prepare its DNA for transfer.
e.g. ColE1
Self-transmissible plasmid
Is both conjugative and mobilizable
Contain tra and mobgene
e.g.F-plasmid
Plasmid

12. Conjugativefunctions are not plasmid specific
E.g. Single cell containing both Fand ColE1
plasmids can transfer ColE1to recipient.
But cell containing only ColE1unable to transfer.

13. Mobilizablefunctions are usually plasmid specific
E.g. Self transmissible plasmid cannot enable a
nonmobilizable plasmid to be transferred.
But transfer can occur when two plasmid recombine
and form a single transferable DNA molecule.

14. Mechanism of Conjugation
1. Formation of specific donor-recipient pairs i.e.
Effective contact
2. Preparation for DNA transfer
3. Actual DNA transfer
4. Formation of replicative functional plasmid in recipient

15. 1. Effective contact
Through pilus(traA)produced by donor in gram
negative bacteria.
(traB, C, E, F, H, K, L, Q, U, V, W)
In gram positive contact is due to mating protein
produced by the recipient (e.g. Streptococcus
faecalis).

16. It is the tipof pili that
make the contact with that of
the recipient.
Donor
Recipient
Pilus

17. 2. Preparation for DNA transfer
Transfer is notthrough the pilus as it is earlier thought.
Route is through the envelope. Initially pairs are
sensitive to shear forces but later stabilized by
unknown process.
Pairs formed due to pili leading to fusing at locally of
envelope between two bacteria. (traG, traN)
(Some proteins can also be transferred with DNA)

18. 3. Actual DNA transfer
a. Origin of transfer
b. Nicking at oriT
traYencode for protein which has got endonuclease activity.
Strand which is denserof two in polyuridylate and polyguanylate cesium
chloride gradientis nicked.
c. Initiation of DNA transfer
Initiation of plasmid is done by traMgene encoded protein.

19. 3. Actual DNA transfer
d. Strand separation and transfer
DNA helicase I product of traI(now considered as traH)
Transfer of ss plasmid DNA is always leaded by 5’
e. Replacement strand synthesis in donor cell
DNAP III required
Rolling circle model of DNA replication

20. 4. Formation of replicative functional plasmid in
recipient (i.e. Functional plasmid formation)
Complementary strand synthesis in recipient cell
Circularization of plasmid DNA
Membrane proteins play important role

22. Some F-plasmid gene and their functions
Sr. Gene Function
1. traA Pilin
2.traB,C,E,F,H,
K,L,Q,U,V,W
For the biosynthesis and assembly of pili
3. traG, N Stabilization of mating pairs
4. traH DNA helicase
5. traJ Regulation of traoperon

23. Some F-plasmid gene and their functions
Sr. Gene Function
6. traM Initiation of plasmid transfer
7. traS, T Inhibition of mating between two donor
8. traY Nicking at oriT
9. oriT Site of nicking
10. oriV Origin of circular DNA replication
11. finO,P Fertility inhibition
12.incB,C,E Plasmid incompatibility

24. F-plasmid
oriT
oriV
tragenes
32 kb
100 kb
Used to initiate
replication for transfer
Used to initiate plasmid
replication
IS elements (insertion
sequences)
Discrete region that has tra
genes (~40 genes)
(Origin of transfer)

25. Conjugative processes
Three main conjugative processes are:
I. F
+
conjugation
II. Hfr conjugation
III. Resistance plasmid conjugation

26. I.F+ Conjugation
Process

27. F+ Conjugation
“Genetic recombination in which there is a transfer of an F+ plasmid
(coding only for a sex pilus) but not chromosomal DNA from a male
donor bacterium to a female recipient bacterium.”
Involves a sex (conjugation) pilus.
Other plasmids present in the cytoplasm of the bacterium, such as
those coding for antibiotic resistance, may also be transferred during
this process.

28. Four stepped F+ Conjugation

29. F
+
F
-
F Pilus Formation
F
FF
F
+

30. II.Hfr Conjugation

31. Hfr Conjugation:
“Genetic recombination in which fragments of
chromosomal DNA from a male donor bacterium are
transferred to a female recipient bacterium following
insertion of an F+ plasmid into the nucleoid of the donor
bacterium.”
Involves a sex (conjugation) pilus.

32. Chromosome Transfer: Formation of Hfr strains
(high frequency recombination)
Depending on
the site of F-
plasmid
integration there
are different Hfr
strains

34. Hfr to F’ Conversion
Integrated F plasmid can
aberrantly excise
Often includes portion of
host chromosome
New plasmid called F’
1 in ten million cell get F’
from Hfr

35. Cell with F’ is partiallydiploidand called a merozygote(very useful for studying genetic
regulation in bacterial systems)

36. III.Resistant Plasmid
Conjugation

37. Resistant plasmid conjugation:
“Genetic recombination in which there is a transfer of an R
plasmid (a plasmid coding for multiple antibiotic resistance
and often a sex pilus) from a male donor bacterium to a female
recipient bacterium.”
Involves a sex (conjugation) pilus.

38. Four Stepped Resistant Plasmid Conjugation

39. DonorRecipientMolecules transferred Product
F
+
F
-
F plasmid F
+
Cell
Hfr F
-
Initiating segment of F
plasmid and variable
quantity of
chromosomal DNA
F
-
with variable
quantity of
chromosomal DNA
F
+
F
-
F
+
plasmid and some
chromosomal genes it
carries with it
F
+
Cell with some
duplicate gene pairs:
one on chromosome,
one on plasmid
Result of selected conjugation

40. Conjugation in Gram positive bacteria
Pili are absent, hence transfer mechanism is different
biochemically as well as genetically
E.g. Streptococcus faecalis
They carry self-transmissible plasmid don't encode
for pilin but for other protein called adhesin

41. Mechanism of conjugation
Recipient cell produce mating protein(analogous to pheromones in insects)
Pheromone signals to donor for synthesis of adhesinthat coats the donor
Adhesin causes Donor-Recipient pairs
Plasmid transferred
Synthesis of pheromone is inhibited

42. References:
Microbial Genetics, Sec. Edi.,
By Maloy, Cronan, Freifelder
Genes VIII
Microbiology, Fifth Edi.,
By Pelczar, Chan, Krieg
http://pcb4522.ifas.ufl.edu

43. Any Questions ..…?

44. Presented By:
Ramane Sangram P.
4673
( Division of VBM )

45. THANKYOU