transformation, conjugation and transduction.pptx

Transformation is the uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule into the recipient chromosome in a heritable form.
In natural transformation the DNA comes from a donor bacterium. The process is random, and any portion of a genome may be transferred between bacteria.
The transformation frequency of very competent cells is around 10-3 for most genera when an excess of DNA is used.

1. Transformation Transformation is the uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule into the recipient chromosome in a heritable form. In natural transformation the DNA comes from a donor bacterium. The process is random, and any portion of a genome may be transferred between bacteria. The transformation frequency of very competent cells is around 10 -3 for most genera when an excess of DNA is used.

2. Competent Cell: Cells that are able to take up the DNA and be transformed. Competency is a complex phenomenon and is dependent on several conditions and increases the efficiency of transformation. In laboratory condition, cells are made competent by treating the cells with chemicals like PEG, CaCl 2 etc are used to increase the porosity of cell membrane. Ultracompetent cells: Highly competent cells.

3. Mechanism of Transformation

4. Transformation is a very difficult process, with very low efficiency . Only some of the DNA gets taken up by the cells which is clearly influenced by the concentration of DNA. After DNA is taken up by the cells, there is two possible fate it could have, DNA is degraded by the cellular mechanism i.e., DNAses present in the cell. Is integrated into the genome via recombination forming stable transformation.

5. Mechanism of Transformation

6. Transformation could also be achieved by the plasmid DNA . In this mode of transformation, DNA taken up by the cell is not degraded. Integration depends on the fact that the plasmid have Insertion sequence (homologous sequence to that of the chromosome). Transformation with a plasmid often is induced artificially in the laboratory.

7. Mechanism of transformation in S. pneumoniae

8. A competent cell binds a double-stranded DNA fragment if the fragment is moderately large; the process is random, and donor fragments compete with each other. The DNA then is cleaved by endonucleases to double stranded fragments about 5 to 15 kilobases in size. DNA uptake requires energy expenditure. One strand is hydrolyzed by an envelope-associated exonuclease during uptake; the other strand associates with small proteins and moves through the plasma membrane. The single-stranded fragment can then align with a homologous region of the genome and be integrated

9. Transformation in Haemophilus influenzae , a gram-negative bacterium, differs from that in S. pneumoniae in several respects. Haemophilus does not produce a competence factor to stimulate the development of competence, and it takes up DNA from only closely related species. Double-stranded DNA, complexed with proteins, is taken in by membrane vesicles. The specificity of Haemophilus transformation is due to a special 11 base pair sequence (5′AAGTGCGGTCA3′) that is repeated over 1,400 times in H. influenzae DNA. DNA must have this sequence to be bound by a competent cell.

10. Conjugation Three types of conjugation: F + × F - conjugation Hfr conjugation. F′ Conjugation.

11. The molecular mechanism of conjugation

12. Conjugation Self- transmisible plasmids and mobilizable plasmids Self-transmissible plasmid coded by tra genes Promiscuous plasmid: interspecies transfer of plasmid

13. Transfer ( tra ) genes eex (entry exclusion): preventing entry of other plasmids with same Tra function oriT gene: origin for transfer Dtr component: “DNA transfer and replication” tra gene protein involved in processing of plasmid DNA to prepare it for transfer Mpf component: “Mating pair formation” Pilus and channel for transfer of plasmid

14. Partial genetic and physical map of F-plasmid

15. The Dtr component Relaxase Specific DNA endonuclease that makes a single strand nick at specific nic site in the ori T sequence On the nick site cause formation of a transesterifiction reaction at 5’ end to one of its Tyrosine Relaxase protein is transferred to recipient cell and along with it the DNA Recyclized DNA after transfer; gets degraded after its function Relaxosome : group of proteins bound to oriT sequnce of plasmid Help bind relaxase , help separate DNA to initiate transfer, help in communication with coupling protein of Mpf system and tell relaxase when to cut; helicase action; Not transferred along with DNA

16. Relaxase action

17. Primase To prime plasmid replication Made in donor cell, not in the recipient Transferred to recipient cell to make more promiscuous and be able to transfer to a wide variety of bacteria Other proteins such as RecA , proteins that form channels in recipient cell membrane The proteins transferred are for docking on the channels and transportation of DNA

18. The Mpf system This system hold donor and recipient together during mating and forms the channel through which protein and DNA are transferred during the mating Also includes system of signaling Dtr system about mating pair formation and initiates the transfer of DNA The pilus 10nm in diameter with central channel; made up of single protein called pilin protein many types found Long, thin, flexible pilus : allows tranfer in liquid medium+aggregation of cells Long rigid pilus /short, thick, rigid pilus : hold mating cells together on solid surface where they are less free to move Some produce both long, flexible and well as short, thick and rigid pilus to make themselves more versatile PASSING OF DNA DURING CONJUGATION THROUGH PILUS SEEMS NOT TO BE TRUE

19. The channel: a pore through which DNA passes during conjugation Little is known of its exact structure and it has escaped detection Coupling protein: bound to membrane channel and passes information to Dtr about contact Coupling proteins ‘dock’ proteins on the membrane channels that are to be trasported

20. Mechanism

21. Mechaism of plasmid mobilization

22. Tri-parental mating form mobilization of plasmid

23. Formation of Hfr Strains

24. Transfer of chromosomal DNA by Hfr Strain

25. Formation of recombination types

26. Creation of prime factor Created by transposition or homologous recombination Recombination between the repeated sequence such as insertion sequence(IS) or genes or rRNA which exists in multiple copies Prime factor can contain essential genes so until the prime factor is within a cell the cell survives and the point when prime factor leaves the cell the cell dies Prime factor contain an entire self-transmissible plasmid, hence the recipient can be donor again Prime factor contains plasmids origin of replication so can replicate in any new bacterium that falls within the plasmid host range

27. Prime factor (F’/R’ etc)

28. Transfer system in Gram positives Plasmid attracting pheromones Pheromones stimulates mating with cells containing a particular plasmid Stimulates expression of tra gene in plasmids of neighboring bacteria, thereby inducing aggregation and mating After plasmid transfer specific pheromone production is stopped but other types of pheromones are produced that will stimulate mating with cells containing other types of plasmid Observed in Bacillus, Streptococcus, Staphylococcus, and Streptomyces

29. Recipient cell

30. Donor

31. Mating induction

32. Plasmid transfer

33. Transduction Genetic recombination in which a DNA fragment is transferred from one bacterium to another by a bacteriophage

34. Bacteriophage Life Cycle Lytic cycle Attachment Penetration Biosynthesis Maturation Release

35. Bacteriophage Life Cycle Lysogenic cycle Attachment Penetration Prophage Cell division Biosynthesis Maturation Release

36. Lysogenic cycle Lysogenic Cycle: Lambda as an example lambda integrase and lambda repressor cI synthesized due to activation of the transcription of their genes by cII . cI repressor turns off phage transcription integrase catalyzes integration of lambda DNA into bacterial chromosome via short sites of homology (site-specific recombination at ATT site) ---- prophage Cro proteins

37. Return to be a killer Prophage: Bacterium is now immune to infection by another phage, because repressor continuously produced ----- new phage DNA can be injected into cell and is circularized but is not transcribed or replicated. Prophage can be excised when host response system to potentially lethal situations: if host DNA damaged one reaction by host cell is to activate a protease protease also cleaves repressor Phage DNA now transcibed including a gene for an enzyme that cuts prophage DNA from bacterial chromosome Lytic cycle can start.

38. Transduction There are two types of transduction: generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle. 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

39. Seven steps in Generalised Transduction 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 or around a plasmid instead of a phage genome by mistake.

40. 4. The bacteriophages are released. 5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

41. 6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium . 7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

42. In general transduction, the fate of DNA molecule transformed via bacteriophage could be different: It may get incorporated into the genome of the bacteria. It may get degraded. DNA is not integrated but often is able to survive and express itself. Abortive transductants are bacteria that contain this nonintegrated, transduced DNA and are partial diploids. Generalized transducing particle or phage and is simply a carrier of genetic information from the original bacterium to another cell.

43. Six steps in Specialised Transduction 1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome . 2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

44. 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. 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.

45. 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.

46. As in general transduction, in special transduction, transformed DNA could have different fate: Crossover to integrate bacterial genes: only bacterial chromosome is transferred via crossover due to homologous sequence present in the DNA. In this type of transduction some section of recipient cell gets replaced by the new DNA. Integration as prophage : DNA, containing both virus and donor DNA gets integrated into the chromosome of recipient cell’s chromosome.