H.Nur Halipçi- conjugation


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  • Prototroph; Any microorganism that can synthesize its nutrients from inorganic material
  • Homolog rekombinasyon: bir bireyin sahip olduğu DNA moleküllerinin veya aynı türe ait bireylerin DNA larının benzerlik gösteren bölgeleri arasında parça hareketleri Tanspozisyon:Genomda farklı gölgelerin arasındaki DNa parça hareketleri
  • H.Nur Halipçi- conjugation

    1. 1. L ecture 19- Conjugation By: HATİCE NUR HALİPÇİ
    2. 2. Bacterial conjugation: is the transfer of genetic material between bacteria through direct cell-to-cell contact, or through a bridge-like connection between the two cells
    3. 3. BACTERIAL CONJUGATION <ul><li>Discovered in 1946 </li></ul><ul><li>by Joshua Lederberg </li></ul><ul><li>and Edward Tatum , </li></ul><ul><li>JOSHUA LEDERBERG EDWARD L. TATUM </li></ul><ul><li>(1925-2008) (1909-1975) </li></ul>
    4. 4. Discovery of conjugation in E. coli by Lederberg and Tatum, 1946 <ul><li>a. Strain A, which is met- and bio-, produces no colonies when plated onto minimal medium. </li></ul><ul><li>b. Strain B, which is thr-, leu-, and thi-, produces no colonies when plated onto minimal medium </li></ul><ul><li>c. A mixture of strains A and B is allowed to grow for a few cell divisions in complete medium and then plated on minimal medium, 1/10,000,000 cells grow into colonies; therefore,a recombinational process is taking place </li></ul><ul><li>d. Cells in the colonies which grew were prototrophs (wild-type) </li></ul>
    5. 5. Davis U tube experiment 1950 Tatum and Lederberg 1947
    6. 6. Davis' U-tube experiment (1950) <ul><li>    1. Cells of strain A were placed on one side of a U-tube and cells of strain B were placed in the other side.  A filter with pore size that allowed liquid but not bacterial cells to pass separated the two sides of the tube </li></ul><ul><li>    2. The medium was pumped back and forth by air pressure/vacuum </li></ul><ul><li>    3. Cells from both sides were plated on minimal medium and none grew </li></ul><ul><li>    4. He concluded that cell-to-cell contact is necessary for genetic recombination to occur </li></ul>
    7. 7. BACTERIAL CONJUGATION <ul><li>Bacterial conjugation is often incorrectly regarded as the bacterial equivalent of sexual reproduction or mating . It is not actually sexual, as it does not involve the fusing of gametes and the creation of a zygote , nor is there equal exchange of genetic material. It is merely the transfer of genetic information from a donor cell to a recipient </li></ul>
    8. 8. The F plasmid is transferred by conjugation between bacteria s 1. Bacterial conjugation : a plasmid genome or host chromosome is transferred from one bacterium to another in a mating process mediated by F plasmid . 2. F-plasmid : an example of an episome in E. coli . 3. Episome : an element that may exist as a free circular plasmid , or that may become integrated into the bacterial chromosome as a liner sequence.
    9. 9. F-plasmid <ul><li>E.coli cells totally lacking the presence of the F factor in any form are called F – cells </li></ul><ul><li>F factor can, however, exist in a cell in three different forms: </li></ul><ul><li>1- Cell containing an autonomously replicating F plasmid are called F + cells </li></ul><ul><li>2-The F factor is able to integrate in to the donor cell chromosome to give rise to an Hfr cell that can efficiently transfer donor cell chromosal DNA to a recipient cell bye conjugation </li></ul>
    10. 10. F-plasmid <ul><li>3- Prime Factor Plasmids that “leave” the genome carrying chromosomal DNA are known as prime factors . </li></ul><ul><li>They “leave” the chromosome by homologous recombination, resulting in a deletion in the chromosome </li></ul>
    11. 11. The F' state and merozygotes <ul><li>a. The F factor can excise from the chromosome and include a part of the bacterial chromosome to become a F‘ </li></ul><ul><li>b. When a F' is mated with a F-, both resultant cells become F' and both cells also contain the bacterial chromosomal segment that was present in the F' </li></ul><ul><li>c.Merozygote is a state when a bacterial cell, is temporarily diploid as result of DNA transfer processes like conjugation </li></ul>
    12. 12. F-plasmid 1. large circular plasmid ( 100 kb ) 2. only 60% (ca. 60 genes ) has been mapped. 3. 32 kb is organized as a unit to transfer its genome to another bacteria ( transfer region or tra genes) 4. two methods of replication: a. oriV as free plasmid (one copy/ bacterial chromosome) b. uses E. coli chromosomal origin when integrated ( oriC ); oriV is suppressed.
    13. 13. F-plasmid oriT oriV tra genes 32 kb 100 kb used to initiate replication for transfer used to initiate plasmid replication IS elements (insertion sequences used in transposition) Discrete region that has transfer genes: tra & trb loci (~40 genes) (Origin of transfer)
    14. 14. F-plasmid IS element F-plasmid E. coli chromosome integrated F-plasmid oriC oriV After integration, F-plasmid replicates as part of host replicon. oriV is suppressed. Hfr cell contains integrated F plasmid. F + cell contains episomal F plasmid
    15. 15. Chromosome Transfer: formation of Hfr strains ( h igh f requency r ecombination) There are two mechanisms of integration: 1. Homologous recombination 2. Transposition Depending on the site of F-plasmid integration there are different Hfr strains
    16. 16. The Sex Pilus -Some E. coli strains contain Fertility plasmids (F + ) - Carries the information required for its own transfer - DNA is NOT transferred through the pilus
    17. 17. F-Pilus 1. Extracellular filament that extends from surface (2-3  m); hair-like -main structural component is a single subunit ( pilin ; coded by traA gene) -tip protein 2. Pilus recognizes various receptors on the host cell. -mating pair formation may occur differently on solid or liquid media
    18. 18. recipient F donor pilus F pili are essential for initiating pairing but are NOT channels for DNA transport
    19. 19. Single stranded genomes are generated for bacterial conjugation 1. F-plasmid is ~100kb & takes about 5 min to transfer. 2. Sequences required for transfer are located within a 33.3-kb transfer region (IncF1) 3. –Pilus synthesis and assembly -Nicking & initiation of transfer -mating pair (pore) formation -transfer of DNA -mating-aggregate stabilization -surface exclusion -regulation
    20. 20. oriT tra M J Y A LEKBPVRC WU N trbCDE traF trbB traH G ST D I /Z finP Transfer genes traJ activator traY/I Transcription unit tra region of the F plasmid Direction of transfer regulation tra & trb loci; ~40 genes Expressed coordinately as a part of single transcription unit traY/traI
    21. 21. F-plasmid 5. F-positive ( F + ) bacteria are able to conjugate (mate) with F-minus ( F - ) bacteria. 6. In its integrated form the F-plasmid may cause some, or all, of the bacterial chromosome to transfer to the F-minus recipient. 7. F-positive bacteria possess pili formed from the pilin protein.
    22. 22. Conjugation <ul><li>tip of the F-pilus makes contact with recipient cell. </li></ul><ul><li>a. pilus is composed of pilin subunits which form a hollow cylinder of 8 nm with 2 nm inner diameter. </li></ul><ul><li>b. If potential recipient is F-positive, no connection is formed due to surface exclusion proteins coded by traS and traT of F-plasmid. </li></ul>
    23. 23. Conjugation 2. Pilus retracts bringing recipient closer for transfer. 3. DNA transferred through channel formed by protein coded by traD gene. TraN and TraG may also participate in pore formation. 4. Transfer begins from oriT which is nicked by TraY/TraI complex at a nic site. (TraI actually nicks) 5. TraY/TraI multimeric complex migrates around circle and unwinds DNA from 5’ end . 1200bp/sec . 6. Only one unit length is transferred.
    24. 24. Overview of Conjugation <ul><li>Plasmids that are both conjugative and mobilizable are termed self-transmissible . Plasmids that are mobilizable but nonconjugative ,are called mob plasmid .They are often efficiently transferred to recipient cells when other plasmids present in the donor cell provide the necessary cell contact functions . </li></ul>2. The transfer systems are encoded by the tra genes (contained on the plasmids themselves).
    25. 25. Overview of Conjugation <ul><li>3. Tra systems are linked to their incompatibility (Inc) group. F-type  IncF, RP4 plasmids  IncP </li></ul><ul><li>4. Plasmids that have tran s fer systems that allow transfer of DNA to unrelated species are known as promiscuous plasmids. IncW plasmids, IncP plasmids, & IncN. </li></ul>
    26. 26. 1. A site on the plasmid, known as the origin of transfer ( oriT ) is nicked by a specific endonuclease (TraI; TraY is also a part of the complex). 2. A pore is formed between the two cells and only ONE strand of DNA is passed through to the other cell ( 5’ end first ). 5’ 3. The single strand in each cell undergoes replication to form double stranded DNA. Only a single unit length of F factor is transferred F  F - F  F  Free 5’ end Mechanism of self-transmissible transfer
    27. 27. 1. The mob plasmid cannot transfer without another plasmid 2. The other plasmid ( helper plasmid ) may or may not be a self-transmissible plasmid but MUST contain tra functions (cell contact, nicking). 3. If the helper plasmid is self-transmissible it may also transfer. tra tra tra tra tra mob helper Mechanism of nonconjugative plasmid mobilization
    28. 28. Major Functions During Transfer (>40 genes) 2. TraI has nuclease & helicase (ATP) activity. Function enhanced by TraY & IHF (integration host factor) TraI is a transferase  covalent attachment of the 5’ end of the DNA to the protein 3. TraD –active transport, binds DNA, ATP/GTP binding sites, necessary for DNA transfer 1. TraY binds near oriT and recruits traI relaxase ,
    29. 29. Surface Exclusion Reduces conjugation among cells carrying closely related plasmids . 1. TraT –outer membrane protein that blocks mating-pair formation 2. TraS –blocks DNA transfer
    30. 30. Chromosome Transfer: Hfr  Recipient cell (Part 1) 1. 2. 3. Free 5’ end Hfr cells F+
    31. 31. Chromosome Transfer: Hfr  Recipient cell (Part 2) 5. 3. 4. oriT region only
    32. 32. Chromosome transfer 1. The transfer process uses the rolling circle method of replication. The complement to the transferred strand is synthesized in the recipient. It takes 100 min to transfer entire chromosome of E. coli. 2 . F-positive strains support high levels of recombination and are described as Hfr strains ( high frequency of recombination ).
    33. 33. 3 . The transfer of the host chromosome is away from the tra region and F-plasmid, except for a small part around oriT . 4 . Typically only relatively short stretches of DNA are transferred & are integrated into the recipient. Chromosome transfer
    34. 34. 5 . Chromosome transfer usually does not result in conversion of recipient cell to F + . 6 . In chromosome transfer, donor DNA integrates into the host genome by recombination or transposition -In plasmid transfer, this does not occur. Chromosome transfer
    35. 35. 7. Bacterial contact usually broken before DNA transfer complete; A gradient of transfer frequencies around the chromosome Chromosome transfer 8. E. coli chromosome as a map divided into 100 minutes; the starting point for the gradient of transfer is different for each Hfr strain; determined by the F factor integration site. 0 &100 50 25 75 E. coli
    36. 36. oriT tra M J Y A LEKBPVRC WU N trbCDE traF trbB traH G ST D I /Z finP Transfer genes traJ activator pilin Surface exclusion Channel Negative regulator transcript traY/traI DNA nicking and unwinding tra region of the F plasmid Senses that mating pair formed Direction of transfer regulation traT -outer membrane protein that blocks mating pair formation. traS -blocks DNA transfer. traI - covalently attaches to 5’ end of DNA & unwinds it finO Helps finP traY - recruits traI to 5’ end of DNA
    37. 37. <ul><ul><ul><ul><ul><li>THANKS.. </li></ul></ul></ul></ul></ul>