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

These slides contain information about bacterial conjugation.

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

2. 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.

3. 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.

4. LEDERBERG AND TATUM EXPIREMENTS (1946)

5. 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 .

6. DAVIS “U” TUBE EXPIREMENTS (1950)

7. 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.

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

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

10. 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.

11. 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.

12. 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+.

13. 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.

14. 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).

15. 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.

16. F- GENETIC CROSSES F+ × F- genetic crosses Hfr × F- genetic crosses F’ × F- genetic crosses

17. 1. F+ × F- genetic crosses

18. 2. Hfr × F- genetic crosses

19. 3. F’ × F- Genetic Crosses

20. REFERENCE https://microbenotes.com/bacterial-conjugation/ https://www.nature.com https://biologydictionary.net

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