Bacterial genetics- gene mapping by recombination

Bacterial Genetics, conjugation, Hfr strains, gene mapping

1. Bacterial gene mapping by Recombination Gurvinder kaur PhD

2. 30-04-2021 2 Bacterial genome Single large circular chromosome – ds DNA which is known as bacterial chromosome or nucleoid . Having contour length of about 1.360 μ m & 20Ǻ long It has 50 or more highly twisted or supercoiled loops and about 4 million nt pairs. Genome is haploid Bacteria reproduce asexually True breeding- i.e. maintain their characteristics from generation to generation

3. 30-04-2021 3 Each bacterial cell contains from 1- 20 much smaller circular DNA molecules known as plasmids or extra chromosomal genetic molecules Contain small number of genes Can incorporate themselves into bacterial chromosome Episome : genetic elements that can exist either as a plasmid or as part of the bacterial chromosome

4. Bacterial growth 4 Bacteria have 4 phases when grown in culture Bacteria can be grown in liquid media and on solid (agar) media

5. 30-04-2021 5 Wild type bacteria are usually prototrophic : can synthesize all necessary compounds. Prototrophs can grow on minimal media. Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used to grow "wild type" microorganisms Prototrophy can be lost by mutations leading to auxotrophy . Auxotrophy is the inability of an organism to synthesize a particular compound required for its growth. Auxotrophs cannot grow on minimal media.

6. 30-04-2021 6

7. Genetic recombination 30-04-2021 7 Microorganisms carry out several types of recombination- General, non-reciprocal & site specific recombination. General recombination-reciprocal exchange between a pair of homologous DNA sequences. It is known as crossing over and is carried out by the products of rec genes such as recA protein which is important for DNA repair. Recombination of genes in bacteria involves transfer of genetic material from one cell to another.

8. 30-04-2021 8 Movement of DNA from a donor bacterium to the recepient can take place in three ways Congugation Transformation transduction

9. Conjugation 30-04-2021 9 Discovered by Joshua Lederberg and Edward Tatum in 1946. Unidirectional transfer of genetic material between donor and recipient bacteria cells by direct contact. Segment of the donor’s chromosome recombines with the homologous recipient chromosome. Recipients containing Donor DNA are called transconjugants .

10. 30-04-2021 10

11. Lederberg’s experiment 30-04-2021 11 They used two strains one that require biotin (Bio - ),phenylalanine ( Phe - ), and cysteine ( Cys -) and other strain requires threonine ( Thr -), leucine ( Leu -) and thiamine( Thi -) for their growth. They mixed these strains and incubate them for several hours & plated on minimal media. Majority of cells failed to multiply, only few of them were able to grow, they assumed that these cells would have been the prototrophs (wild type) will all the genes dominant. Thus the chromosomes of two auxotrophs were able to associate and under go recombination.

12. Evidence of Genetic recombination in bacteria 30-04-2021 12 Spontaneous mutation alone could not explain why the mixture of cells could survive in minimal medium Cells that survived must have acquired genes from the other mutant strain Even though new mutations are a major source of genetic variation in bacteria, genetic recombination adds more diversity. The exchange of DNA strands between duplex DNA molecules is an event central to mechanisms of genetic recombination.

13. Recombination by conjugation 13 William hayes , Cavallisforza and Jacob and Wollman (1958), conculded that bacteria exhibit sexual reproduction and behave like male and female gametes of higher forms It is the one way DNA transfer: F+ male or donor strain- possess the fertility factor F- female or recepient strain- without the F factor. Mechanism Donor extends sex pilli to recipient Sex pilus retracts, pulling cells together DNA transferred through cytoplasmic bridge or conjugation tube . Conjugation begins when Fplasmid is nicked at the origin, and a single strand is transferred using the rolling circle mechanism. when the transfer is complete, both cells are F+ double stranded.

14. 30-04-2021 14

15. 30-04-2021 15 Conjugation Coupling factor Coupling factor F+ x F- conjugation

16. 30-04-2021 16 Conjugation contd … In the donor cells, the F factor DNA is replicated to become dstranded . In the recipient cell, relaxase joins the ends of the T-DNA strand. It is then replicated to become double stranded. F+cell F+cell

17. 30-04-2021 17 Hfr strain In Hfr conjugation, donor cells transfers the bacterial gene with great efficiency into the recepient . Donor exhibit high frequency of gene transfer and carry integrated F factor.

18. 30-04-2021 18 Hfr conjugation The recipient F- cell will remain F- after Hfr conjugation as complete F factor is not transferred

19. 30-04-2021 19

20. 30-04-2021 20

21. 30-04-2021 21

22. 30-04-2021 22

23. 30-04-2021 23

24. 30-04-2021 24

25. 30-04-2021 25

26. 30-04-2021 26

27. 30-04-2021 27

28. 30-04-2021 28

29. 30-04-2021 29

30. 30-04-2021 30

31. 30-04-2021 31

32. 30-04-2021 Free template from www.brainybetty.com 32

33. 30-04-2021 Free template from www.brainybetty.com 33

34. 30-04-2021 Free template from www.brainybetty.com 34

35. 30-04-2021 Free template from www.brainybetty.com 35

36. 30-04-2021 Free template from www.brainybetty.com 36

37. 30-04-2021 Free template from www.brainybetty.com 37