Quorum Sensing

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  1. *What is quorum sensing?
    cell density dependent regulation

    quorum - minimum number required to conduct business
  2. initial discovery of quorum sensing
    first observed as control of bioluminescence in bacteria

    • marine bacteria, associated with fish - vibrio fischeri, photobacterium
    • terrestrial bacteria - xenorhabdus luminescens
  3. vibria fischeri and induction of luminescence
    • free living in ocean do not give off light, cell density 102 cells/ml
    • at high densities such as in light organs do luminesce, cell density 1010 - 1011 cells/ml

    reaction is catalyzed by luciferase - luxA and luxB genes encode the alpha and beta subunit of heterodimer
  4. Vibrio fischeri symbiosis with Eupryma scolopes
    • juvenile squid becomes infected with V. fischeri - species specific
    • light organ matures, has developed three specialized structures to manipulate light produced by the bacteria
    • squid is nocturnal forager - luminescing bacteria erase its shadow cast by moon, protecting it from predators below
    • squid provides stable source of nutrients for bacteria
  5. Is something produced by the squired required?
    • isolate V. fischeri from squid light organ, obtain pure culture
    • can it luminescence? Yes, but only when culture is in exponential phase

    No, squid product is not required, however bacteria must be in late exponential phase
  6. Does this induction of luminescence require translation and/or transcription?
    • chloramphenical - inhibitor of protein synthesis
    • rifampin - inhibitor of transcription

    yes, luminescence is transcriptionally regulated
  7. What is controlling this transcriptional regulation?
    • perform add-back experiments
    • Image Upload 1
    • spin down cells and pour off medium
    • add cells from a to medium a (control) - no light
    • add cells from a to medium b - light
    • add cells from b to medium a - no light
    • add cells from b to medium b (control) - light
    • observe luminescence over time

    something in cell-free medium b induces luminescence
  8. isolation of autoinducer molecule
    • cell free medium at exponential phase (V. fischeri are producing light) ->
    • extract with ethyl acetate ->
    • separate using chromatography (TLC and HPLC) ->
    • get very pure compound, identify by NMR, MS, UV spectra ->
    • test that is has the expected biological activity
  9. V. fischeri autoinducer
    • N-3-(oxohexanoyl) homoserine lactone
    • induces bioluminescence
    • Image Upload 2
  10. How is cell density sensed and this signal transduced resulting in bioluminescence (or other AI-dependent phenotype)?
    • autoinducer acts as a diffusible signal, indicator of cell density
    • threshold levels of 5-10 nM autoinducer are requried for response via the regulator LuxR that activates the lux operon
  11. *How does Acyl-HSL binding to LuxR enhance transcription?
    LuxR monomer does not bind lux box

    • when sufficient Acyl HSL is present:
    • 1. Acyl-HSL binds LuxR resulting in conformational change
    • 2. LuxR multimerization domain is exposed resulting in formation of a liganded LuxR dimer
    • 3. The liganded dimer then binds the lux box
    • 4. Transcription of the target genes occurs
  12. *quorum sensing in gram negative bacteria
    • *many use Acyl HSL as autoinducers
    • bacteria can produce multiple Acyl HSLs
    • mediate population-sensitive behavior
  13. *quorum sensing in gram positive bacteria
    • *autoinducers are autoinducer peptides:
    • vary in amino acids
    • often contain side-chain modifications

    • 1. precursor protein is translated and cleaved
    • 2. AIP is secreted via ABC transporter
    • 3. minimal stimulatory extracellular concentration of AIP accumulates
    • 4. two component sensor kinase/ response regulator detects and responds to AIPs - binding is highly sequence specific
    • 5. phosphorylated response regulator activates transcription of target genes

    Image Upload 3
  14. S. aureus autoinducer peptide structure
    Image Upload 4
  15. specificity of S. aureus quorum sensing
    • AIP interacts with N-terminal of receptor kinase
    • - specificity conferred by variations in this region
    • hypervariable regions of receptor kinase, precursor protein and ABC transporter coevolve
  16. AIP type-specific inhibition of quorum sensing
    AIPs inhibit quorum sensing in strains and species of other AIP types

    • results in:
    • isolation of populations
    • act as a major determinant of strain and species divergence
  17. *bacterial communication between species
    • *autoinducer-2 are furans - universal autoinducer
    • AI-2 synthase produced and control gene expression in a number of bacteria
    • hosts like plants can synthesize something for autoinducer inference
    • target for antibiotics?
  18. *What processes are mediated by quorum sensing?
    • bioluminescence
    • expression of virulence genes
    • competence/sporulation
Card Set
Quorum Sensing
general microbiology midterm 3
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