5. Bacterial Genetics II Genetic Exchange

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cornpops
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101943
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5. Bacterial Genetics II Genetic Exchange
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2011-10-04 02:03:09
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PMB 112 midterm1
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general microbiology midterm 1
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  1. horizontal gene transfer
    • natural genetic exchange among bacterial strains/species
    • evolutionary force
    • how antibiotic resistance spreads
  2. uses of genetic exchange
    • to identify affected genes in mutants
    • to map genes
    • to engineer bacteria to have desired properties
  3. transformation
    transfer of genetic material between bacteria
  4. Griffith experiment
    • demonstrating transformation
    • infected mice with live R (rough, avirulent) cells and heat-killed S (smooth, virulent) cells - killed mice
    • demonstrated that something could transfer the properties (genes) of dead S cells to living R cells
  5. Avery 1944 paper
    • showed that the transforming principle was DNA
    • used anti-R serum - can tell if cells are R strains because clump and fall to the bottom
    • R36A strain - if strain reverts cannot tell if it has undergone transformation
    • did chemical analysis of transforming substance - ratios matched with DNA
    • looked at which enzymes depolymerized DNA and inactivated the transformation substance, measured viscosity
    • differential heat inactivation of enzymes - matched depolymerase activity on DNA
    • titration - find minimum amount necessary for transformation
  6. steps of natural transformation
    • 1) free dsDNA is bound to to a membrane-bound DNA binding protein
    • 2) one strand is taken into the cell while the other is degraded by a cell surface nuclease
    • 3) RecA mediates homologous recombination between foreign DNA and host genome

    recombination must occur or new DNA is not replicated and lost
  7. homologous recombination
    • physical exchange between two highly similar DNA molecules
    • can't integrate entirely foreign DNA into bacterial chromosome - not homologous, more likely to be safe if similar
    • host proteins RecA, RecBCD, RuvABC mediate the process
    • enzymes are not involved in transposition
    • if one circle has single recombination event with another circle - bigger circle, cointegrate
    • if linear piece undergoes two recombination events with a circle - only genes between two crossovers are exchanged
  8. competence
    ability of cells to take up free DNA
  9. how to induce artificial competence
    • incubating cells with high concentrations of Ca++ or by electroporation - increase cell permeability and allow uptake of dsDNA
    • plasmids can be introduced
  10. plasmid
    • circular genetic element that replicates independently of the chromosome
    • does not contain any essential genes
    • relatively small compared to a chromosome
    • replicated by normal cellular machinery
    • have specific host ranges - which bacteria they can replicate in
    • ubiquitous in nature, many have been modified to express foreign genes
  11. bacterial conjugation
    • DNA transfer that requires cell-cell contact
    • plasmid encoded mechanism - transfer copies of themselves into new host cells
    • can be used for genetic elements that cannot transfer themselves
  12. Steps of F plasmid transfer from F+ cell donor to F- cell recipient
    • Pilus connects cells and then retracts
    • F plasmid nicked at origin of transfer
    • one strand of plasmid is transferred into the recipient cell
    • replication is occurring in both cells so they both end up with a complete copy of plasmid
    • cells separate
    • creates two F+
  13. F plasmid
    tra region = region contains genes necessary for plasmid transfer, transferred last

    oriT= origin of transfer, where plasmid mobilization starts

    green = replication origin, genes for plasmid segregation to daughter cells

    yellow = transposons and insertion sequences present in the F plasmid, which can mediate integration into host genome
  14. formation of Hfr strain
    • insertion sequences can be used as a site for homologous recombination between F plasmid and a bacterial chromosome
    • results in Hfr state in which chromosomal genes can be transferred to other cells by conjugation
  15. transfer of chromosomal genes by an Hfr strain
    • transfer proceeds in a fixed direction from oriT
    • tra region is almost never transferred - recipient stays F-
  16. using Hfr strains to map the E.coli genome
    • in Hfr strain, integrated plasmid cannot leave the chromosome
    • F plasmid can insert at various sites in either orientation - creates different Hfr strains, which are then permanent
    • frequency of transfer = 1/distance from F plasmid insertion (genes closest to plasmid with be transferred with highest frequency)
  17. How do we detect conjugation?
    • choose donor and recipient strains so that you can select and screen for recipient colonies with specific phenotypes
    • example - use antibiotic resistance on all plates and different minimal medium plates
  18. interrupted mating experiments for chromosome mapping
    • mix Hfr donor + with recipient -
    • allow mating to occur for various amounts of time, then separate mating pairs in a blender
    • plate on conditions so that only recombinants grow
    • plate to test for other alleles individually
    • calculate frequencies of transfer of each allele
    • significance = were able to map E.coli showing that it was circular, showed genes were linked to each other
  19. generalized transduction
    • bacteriophage-mediated exchange of DNA from bacterial chromosomes
    • when bacterial cell is infected with phage lytic cycle may occur - small fraction of phage accidentally package chromosomal DNA from host instead of a phage genome
    • results in transducing particles = contain fragments of host genome rather than phage genome
    • not all phage perform - some only package DNA
    • with a specific sequence, wouldn't package a random piece
    • after infection of new cell, fragments can recombine into recipient chromosome to yield tranductants
    • can use to transfer mutations from one strain background to another
  20. cotransduction frequency
    • measure of how close two genes are to each other on the chromosome
    • can map genes if they are close enough to be carried on the same piece of DNA packed into a phage
  21. how to combine two mutations in the same strain background
    • infect transposon strain with phage and harvest all phage after cell lysis
    • tiny fraction will contain genomic DNA with the transposon and neighboring chromosomal DNA
    • infect point mutant strain with phage from transposon strain
    • plate cells on medium containing selective marker
    • can now investigate consequences of having both mutations in the same cell

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