Conjugation: Discovery, F+, F- and Hfr conjugation, F- genetic crosses

CONJUGATION:
DISCOVERY, F+, F-, Hfr CELLS
AND
F- GENETIC CROSSES
SIVASANGARI SHANMUGAM

WHAT IS?
 Conjugation is the transfer of genetic material between the bacterial
cell by direct contact/by a bridge like connection between the two.
 Physical contact is required.
 Bacterial conjugation is also known as type IV secretion system.
 Conjugation occurs in and between many species of bacteria,
including gram negative, gram positive bacteria, and even occurs
between bacteria and plants.
 Although numerous examples of conjugative plasmid exist,
conjugation involving the F plasmid is most common.

DISCOVERY
 Discovered by Joshua Lederberg & Edward Tatum in 1946.
 They experimented with two auxotrophic strains of E.Coli
K12.
 Denoted by Strain 1 & Strain 2.
 The evidence for cell to cell contact was provided by Bernard
Davis in 1950.

LEDERBERG AND TATUM
EXPIREMENTS (1946)

 Discovered by Joshua Lederberg & Edward Tatum in 1946.
 They experimented with two auxotrophic strains of E.Coli
K12.
 Denoted by Strain 1 & Strain 2.
 Strain A and Strain B were plated on minimal medium and
incubated at over night, no growth observed.
 Also Strain A and Strain B were mixed together and when
plated on minimal medium resulted in prototrophs.

DAVIS “U” TUBE EXPIREMENTS (1950)

 The evidence for cell to cell contact was provided by Bernard
Davis in 1950.
 The arms of the U tube are separated by a filter.
 On the right side is medium containing auxotrophic strain A
while on the left side is medium containing auxotrophic strain
B.
 The filter allows only the medium but not allows the cell on
either side.
 When culture was plated from both sides on minimal medium,
no prototrophs growth was observed as in Lederberg and
Tatum’ s experiments.

AUXOTROPHS & PROTOTROPHS
1. Auxotrophs
Mutant organisms.
Need additional supplements for their growth.
Need nutrients (AA).
Unable to synthesis themselves.

2. Prototrophs
Wild type organisms.
Which grows in Minimal Medium.

MECHANISM OF F+ AND F-
CONJUGATION
 Donor cell produce a pilus.
 Pilus attaches to recipient cell and brings the two cells
together.
 Only single standard of plasmid is then transferred to the
recipient cell.
 Bothe the cells synthesis a complementary strands to produce
double stranded circular plasmid and also produce pilus.
 Both cells are now viable donor.

 F plasmid contains tra locus, which includes the pilin. This
gene, along with some regulatory proteins results in the
formation of pili on the F+cell surface.
 The protein present in the pili attach themselves on the F- cell
surface.
 The pilli responsible for making contact between the cells, but
the transfer of plasmid doesn’t occur through the pili.
 The traD enzyme, located at the base of the pilus, initiates
membrane fusion.

 Once the conjugation is initiated, enzyme relaxase creates a
nick in the conjugative plasmid at the oriT.
 The nicked strand then unwinds and is transferred to the
recipient cell in the 5’-3’ direction.
 The complementary strand is synthesized in both cells; thus,
donor and recipient are F+.

Hfr CONJUGATION
 High frequency recombination (Hfr).
 In the 1950’s, Luca Cavalli-Sforza discovered a strain of E.coli that was
very efficient at transferring chromosomal DNA.
 He designated this strain as Hfr.
 Hfr strain is derived from F+ strains.

 When F plasmid integrated with chromosomal DNA Such bacteria is
known as Hfr bacteria.
 In the conjugation between Hfr cells and F- cells, Hfr is very high but
frequency of transfer of whole F- factor is very low.
 Hfr cells is donor and F- cell act as recipient.
 F factor makes sex pilus that joins donor and recipient.
 F factor opens as replication origin then one strand is cut down.
 Now 5’ end of the strand enters into recipient cell through conjugation tube
(Pilus).

 5’ end enters first into recipient cell but the portion situated at 3’ end enters
only when whole chromosomal DNA enters into the recipient cell.
 To transfer whole chromosomal DNA, it takes 100mns in E.coli.
 In most of the cases, sex pilus breaks before transfer of whole
chromosomal DNA takes place. So, frequency of transfer of whole F-
factor is very low.
 After the cross between Hfr cell and F- cell, recipient cell remains
recipient.
 In this conjugation, chromosomal DNA is always transfer from donor to
recipient cell together with portion of F- factor. So, frequency of
recombination is high.

F- GENETIC CROSSES
1. F+ × F- genetic
crosses
2. Hfr × F- genetic
crosses
3. F’ × F- genetic
crosses

REFERENCE
1. https://microbenotes.com/bacterial-conjugation/
2. https://www.nature.com
3. https://biologydictionary.net

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