8. Bacterial Cell Division

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  1. homologs of cytoskeletal elements found in eukaryotes
    • actin = MreB, Mbl
    • tubulin = FtsZ
    • intermediate filaments = CreS
  2. MreB
    • mutants lose their characteristic rod shapes and lyse - required for stable cell wall
    • MreB polymerizes into filaments that resemble actin filaments in vitro
    • GFP-MreB and GFP-Mbl assemble into dynamic helical structures that form bands along inside of cell
  3. MreBCD complex
    • experiments using immunoflouresence without MreC or MreB show PBP2 mislocalized and can't make normal lateral walls - cells lose their shape and lyse
    • positions the PG synthesis machinery along lateral cell walls
    • Mre B, C, and D are expressed together in an operon
    • enzymes required for PG synthesis form complex with Mre B, C, and D, are positioned at cell membrane for efficient cell wall synthesis
  4. crescentin (CreS)
    homolog of intermediate filament proteins that are responsible for curved shape of Caulobacter and other cells - mutants are straight
  5. FtsZ
    • fission temperature sensitive screen
    • forms a ring at the site of cell division between the two replicated chromosomes - constriction drives cell division
    • FtsZ is the assembly site for the divisome = large protein complex that spans the cytoplasmic membrane and synthesizes the septal cell wall
    • most subunits are essential for viability
  6. minicell mutants
    all single mutants and combinations of minCDE cause the minicell phenotype except minE which blocks division
  7. MinD and MinC GFP time-lapse microscopy
    • rapid pole-to-pole oscillation of MinD and MinC
    • MinC and MinD form a tube on the cytoplasmic membrane at one end of the cell then migrate to the opposite pole
    • rarely found at the midcell, near the division plane
  8. MinE GFP time-lapse microscopy
    • MinE forms a ring at the medial edge of the MinCD polar zone
    • the E ring moves toward the pole containing MinCD
    • as MinE moves toward the pole, MinCD are released
    • MinC and MinD migrate to the opposite pole and set up a new polar zone
  9. mechanism for Min oscillation
    • 1) MinC directly inhibits FtsZ polymerization
    • 2) MinC binds to MinD
    • 3) MinD-ATP assembles on the cytoplasmic membrane and carries MinC, When MinD hydrolyzes ATP, it is released from the membrane
    • 4) MinE binds to the medial edge of the MinCD polar zone MinE stimulates the ATPase activity of MinD, which releases MinD and MinC from the membrane
    • 4) In the cytoplasm, MinD exchanges ADP for
    • ATP and reassembles with MinC away from MinE
    • 5) Net result is that MinC, the FtsZ inhibitor, spends
    • most of its time at the cell poles and very little time at
    • the midcell
  10. nucleoid occlusion
    • even when Min system is absent, division doesn't occur in areas containing DNA
    • minicells lack DNA while the larger cell receives two chromosomes
    • hypothesis is that nucleoid gives off sigmal preventing FtsZ ring formation nearby
  11. de Boer 1989 paper
    • experiments done in a ΔminCDE background use a loss-of-function strategy - see if restoring some of genes can rescue minicell phenotype
    • results suggest all three are needed for correct placement of division septum and minCD are required to inhibit the formation of the division septum

    experiments in WT background use overexpression to probe function

    expression of MinC and MinD in ΔminCDE strain results in inability to form a septum, expression of either alone does not change phenotype - both function together to inhibit septum formation

    overexpressing minE is epitstatic over overexpressing minCD

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8. Bacterial Cell Division
2011-10-04 21:16:36
PMB 112 midterm1

general microbiology midterm 1
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