h.nur halipçi- conjugation

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  • 1.L ecture19- Conjugation By: HATCE NUR HALP

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. BACTERIAL CONJUGATION

  • Discovered in 1946
  • byJoshua Lederberg
  • andEdward Tatum ,
  • JOSHUA LEDERBERGEDWARD L. TATUM
  • (1925-2008)(1909-1975)

4. Discovery of conjugation inE. coliby Lederberg and Tatum, 1946

  • a. Strain A, which is met- and bio-, produces no colonies when plated onto minimal medium.
  • b. Strain B, which is thr-, leu-, and thi-, produces no colonies when plated onto minimal medium
  • 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
  • d. Cells in the colonies which grew were prototrophs (wild-type)

5. Davis U tube experiment 1950 Tatum and Lederberg 1947 6. Davis' U-tube experiment (1950)

  • 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
  • 2. The medium was pumped back and forth by air pressure/vacuum
  • 3. Cells from both sides were plated on minimal medium and none grew
  • 4. He concluded that cell-to-cell contact is necessary for genetic recombination to occur

7. BACTERIAL CONJUGATION

  • Bacterial conjugation is often incorrectly regarded as thebacterialequivalent ofsexual reproductionormating . It is not actually sexual, as it does not involve the fusing ofgametesand the creation of azygote , nor is there equal exchange of genetic material. It is merely the transfer of genetic information from a donor cell to a recipient

8. The F plasmid is transferred byconjugation between bacteria s 1.Bacterial conjugation : aplasmid genomeorhost chromosomeis transferred from one bacterium to another in amatingprocess mediated byF plasmid . 2.F-plasmid : an example of an episome inE. coli . 3.Episome : an element that may exist as a freecircular plasmid , or that may becomeintegratedinto the bacterial chromosome as a liner sequence. 9. F-plasmid

  • E.coli cells totally lacking the presence of the F factor in any form are calledF cells
  • F factor can, however, exist in a cell in three different forms:
  • 1- Cell containing an autonomously replicating F plasmid are calledF + cells
  • 2-The F factor is able to integrate in to the donor cell chromosome to give rise to anHfr cellthat can efficiently transfer donor cell chromosal DNA to a recipient cell bye conjugation

10. F-plasmid

  • 3-Prime Factor Plasmids that leave the genome carrying chromosomal DNA are known asprime factors .
  • They leave the chromosome by homologous recombination, resulting in a deletion in the chromosome

11. The F' state and merozygotes

  • a. The F factor can excise from the chromosome and include a part of the bacterial chromosome to become a F
  • 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'
  • c.Merozygoteis a state when a bacterial cell, is temporarilydiploidas result of DNA transfer processes likeconjugation

12. F-plasmid 1. large circular plasmid ( 100 kb ) 2. only 60% (ca.60 genes ) has been mapped. 3.32 kbis organized as a unit to transfer its genome to another bacteria ( transfer regionortragenes) 4.twomethods of replication: a.oriVas free plasmid (one copy/ bacterial chromosome) b. usesE. colichromosomal origin when integrated ( oriC );oriVis suppressed. 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&trbloci (~40 genes) (Origin of transfer) 14. F-plasmid IS element F-plasmid E. colichromosome integrated F-plasmid oriC oriV After integration, F-plasmid replicates as part of host replicon.oriVis suppressed. Hfr cellcontains integrated F plasmid. F +cellcontains episomal F plasmid 15. Chromosome Transfer: formation ofHfrstrains ( h ighf requencyr 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. The Sex Pilus -SomeE. colistrains contain Fertilityplasmids(F + ) - Carries the information required for itsowntransfer - DNA is NOT transferred through the pilus 17. F-Pilus 1. Extracellular filament that extends from surface (2-3 m); hair-like -main structural component is a single subunit ( pilin ; coded bytraA gene)-tip protein 2. Pilus recognizes various receptors on the host cell. -mating pair formation may occur differently on solid or liquid media 18. recipient F donor pilus F pili are essential for initiating pairing but areNOTchannels for DNA transport 19. Single stranded genomes are generated for bacterial conjugation 1. F-plasmid is ~100kb & takes about5 minto 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. oriTtra M J Y A LEKBPVRC WU NtrbCDE traF trbB traH GST D I /Z finP Transfer genes traJ activator traY/I Transcription unit traregion of the F plasmid Direction of transfer regulation tra&trbloci; ~40 genes Expressed coordinately as a part of single transcription unittraY/traI 21. F-plasmid 5.F-positive( F + )bacteria are able toconjugate(mate) withF-minus( F - ) bacteria. 6. In itsintegratedform the F-plasmid may cause some, or all, of thebacterial chromosome to transferto the F-minus recipient. 7. F-positive bacteria possesspiliformed from thepilinprotein. 22. Conjugation

  • tip of the F-pilus makes contact with recipient cell.
  • a. pilus is composed of pilin subunits which form a hollow cylinder of 8 nm with 2 nm inner diameter.
  • b. If potential recipient is F-positive, no connection is formed due tosurface exclusion proteinscoded bytraSandtraTof F-plasmid.

23. Conjugation 2.Pilus retractsbringing recipient closer for transfer. 3.DNA transferredthroughchannelformed by protein coded bytraDgene. TraN and TraG may also participate in pore formation. 4. Transfer begins fromoriTwhich is nicked byTraY/TraIcomplex at anicsite. (TraI actually nicks)5.TraY/TraI multimericcomplex migrates around circle andunwinds DNAfrom 5 end .1200bp/sec . 6.Onlyone unitlength is transferred. 24. Overview of Conjugation

  • Plasmidsthat are both conjugative and mobilizablearetermed self-transmissible .Plasmids that are mobilizable but nonconjugative ,are calledmob plasmid.They are often efficiently transferred to recipient cells when other plasmids present in the donor cell provide the necessary cell contact functions .

2. The transfer systems are encoded by thetra genes (contained on the plasmids themselves). 25. Overview of Conjugation

  • 3. Tra systems are linked to their incompatibility (Inc) group. F-typeIncF, RP4 plasmidsIncP
  • 4. Plasmids that have tran s fer systems that allow transfer of DNA to unrelated species are known aspromiscuousplasmids. IncW plasmids, IncP plasmids, & IncN.

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. Aporeis formed between the two cells and onlyONE 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 ofself-transmissibletransfer 27. 1. Themobplasmid cannot transfer without another plasmid 2. The other plasmid ( helper plasmid )may or may notbe a self-transmissible plasmid butMUSTcontaintrafunctions (cell contact, nicking). 3. If the helper plasmid is self-transmissible it may also transfer. tra tra tra tra tra mob helper Mechanism ofnonconjugativeplasmid mobilization 28. Major Functions During Transfer (>40 genes) 2.TraIhas nuclease & helicase (ATP) activity. Function enhanced by TraY & IHF (integration host factor) TraI is a transferasecovalent attachment of the 5 end of the DNA to the protein 3.TraDactive transport, binds DNA, ATP/GTP binding sites,necessary for DNA transfer 1.TraYbinds near oriT and recruits traI relaxase , 29. Surface Exclusion Reduces conjugation among cells carrying closely related plasmids . 1.TraTouter membrane protein that blocks mating-pair formation 2.TraSblocks DNA transfer 30. Chromosome Transfer: HfrRecipient cell (Part 1) 1. 2. 3. Free 5 end Hfr cells F+ 31. Chromosome Transfer: HfrRecipient cell (Part 2) 5. 3. 4. oriTregion only 32. Chromosome transfer 1. Thetransfer processuses therolling circle method of replication.The complement to the transferred strand is synthesized in the recipient.It takes100 minto transfer entire chromosome ofE. coli. 2 . F-positive strains supporthigh levels of recombinationand are described asHfrstrains ( high frequency of recombination ). 33. 3 . Thetransfer of the host chromosomeisawayfrom thetraregion and F-plasmid, except for a