quorum sensing

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Presented By, Miss. Harshada M.
SalviB.E.Biotechnology
Mr. Pranav H. Nakhate B.E.Biotechnology
QUORUM SENSING

• Quorum-sensing
• The phenomenon of microbial communication

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Points To Be Discussed
QUORUM SENSING
Bio?lm- a cell-to-cell communication
Quorum Sensing
Bioluminescence- promising application of quorum
sensing
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Bio?lm- a cell-to-cell communication
QUORUM SENSING

• Bio?lm formation .
• cell-to-cell communication -role in biofilm
  formation.
• A Biofilm disadvantageous to humans.
• Eg.
• -P. aeruginosa ,
• -C. albicans ,
• -Haemophilus influenzae .

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QUORUM SENSING
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Inhibition of Biofilm

• Very hard to remove the biofilm , because of its
  physical chemical properties.
• Biofouling of the MBR .
• Effectivity of farnesol over C. albicans bio?lm
  (Ramage et al.)
• Case study by Martinelli et al. (2004).

QUORUM SENSING
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Inhibition of QS by chemical compounds
QUORUM SENSING

• Used Bacterial mutant strain Chromobacterium
  violaceum CV026 .
• Violacein synthesis by bacteria.
• Bioassay -
• Media used- 0.5 yeast extract ,
• -1 tryptone.
• Material used - ?lter paper (0.22-mm),
• - Seawater,
• - QS blockers
  (FUR1,FUR2)
• - spectrophotometer.

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• Concentration of N-hexanoyl homo serine lactones
  (HHL) - 3.7-10.8 M
• Incubation of 270C for 16 hr.
• No QS Blocker HHL Positive control.
• No QS Blocker No HHL Negative control.
• Turbidity measurance at 660 nm.

QUORUM SENSING
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Result
QUORUM SENSING

• Inhibited the violacein in the presence of HHL.
• FUR1 and FUR2 inhibited bacterial QS at
  concentrations of 10-310-5 M.
• Violacein formation reduced up to 90.
• Result shows QS blockers interfere with the
  signalling system.
• signalling system known as QUORUM SENSING.

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QUORUM SENSING
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• Bacteria are single cell organisms with no
  nervous system or brain.
• Environmental conditions change rapidly, bacteria
  need to respond quickly in order to survive.
• So how do individual bacterial cells,"decide
  between different physiological processes living
  as part of a complex community called a biofilm
  ???
• Quorum Sensing (QS) has been found to play a role
  in P. aeruginosa bio?lm formation.

QUORUM SENSING
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QUORUM SENSING

• SO,
• What actually is
• QUORUM SENSING......????

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Quorum Sensing a cell-to-cell communication
QUORUM SENSING

• way of exchange information through
  communication molecule.
• Communication molecules refers to Small
  Molecules (SMs) or specifically, Autoinducers.
• A phenomena, to regulate phenotypes.
• This type of bacterial communication is achieved
  only at higher cell densities.

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Density dependent QS
QUORUM SENSING
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QS Signal Mechanism
QUORUM SENSING

• Involves subsequent interaction of the signal
  with an intracellular effectors.

Cell surface receptors
Signal molecule
Secondary messengers
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QUORUM SENSING

• Bacteria constantly produce and secrete certain
  signaling molecules, called as a auto-inducers or
  pheromones.
• Density dependent phenomena.
• positive feedback loop.

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Q. S. Signal molecules
QUORUM SENSING

• Two main categories-
• (1) Amino Acids and Short Peptide Derivatives,
• (2) Fatty Acid Derivatives, called Homoserine
  Lactones (HSLs).

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Acyl-homoserine lactones (AHLs)
QUORUM SENSING

• Mediate quorum sensing in Gram-negative bacteria.
• induce the synthesis of compounds interacting
  with the host organism.
• several types depending on their length of acyl
  side chain.
• many bacterial species can produce more than one
  type of Acyl HSL.

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The peptide pheromone
QUORUM SENSING

• These are small peptides, regulate gene
  expression in Gram-positive.
• Interact with membrane bound histidine kinase
  receptor.

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Bioluminescence- promising application of quorum
sensing
QUORUM SENSING

• Most studied example is V. harveyi .
• commonly forms symbiotic relationships with some
  fishes.
• These marine animals carry a specialized organ
  called the light organ, in which bacteria like V.
  harveyi are housed.
• FMNNADH H FMN REDUCTASE FMNH 2 NAD
• FMNH 2 RCHO 0 2 LUCIFERASE FMN RCOOH
  
• 
  H 2O LIGHT.

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Isolation of bioluminescence bacteria its
application in toxicity detection.
QUORUM SENSING

• Work has been done in in KIT College of
  engineering, biotechnology department,
  Microbiology laboratory.
• Following objectives are achieved-
• 1) Isolation of bacteria from environment
• 2) Enhancement in the natural bioluminescence
• 3) Study of effect of toxic chemicals on
  bioluminescence

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Isolation of bioluminescent bacteria from
environment-
QUORUM SENSING

• Media peptone (5g/l), yeast extract (2g/l),
  agar-agar (15g/l)
• 75 sea water 25 D/W
• pH - 7
• Temp.- 220 C-250 C for 24 hours
• Gram nature and motility

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QUORUM SENSING
In Dark
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Enhancement in the natural bioluminescence
QUORUM SENSING
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Effect of Autoinducer
QUORUM SENSING

• 200 ml optimized sea water media in 2 flask
  autoclaved.
• 2 innoculum at 22-25 0 C for 18 hrs.
  
• 1 flask- centrifuged at 1000 rpm for 10
  min kept at RT.
• Supernatant added in another flask.
  Luminescence observed.
• 
  

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Study of toxic chemicals
QUORUM SENSING

• 100 ml optimized sea water.

2 innoculum at 22-25 0 C for 18 hrs.
40 ml crude autoinducer( isolated.)
10 min. incubation
0.002 gm/ml CuSO4.5H2O.
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QUORUM SENSING

• C1V1 C2V2
• C1 0.002 gm/ml.
• V10.30 ml.
• V26 ml.
• C2 ?
• Hence, C20.1 gm/lit. Which is EC100 value,
• Hence, EC50 value 0.05 gm/ lit.

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Applications-
QUORUM SENSING

• Gene therapy-to check protein production.
• Cancer cells , tissue damage detection.
• Inhibition of biofilm development by RNA III
  Inhibiting Peptide (RIP).
• SEWAGE SLUDGE treatment.

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Future Prospectus
QUORUM SENSING

• LUMINOMETER-an instrument for studying
  toxicity-bioluminescence relationship.
• Development of a kit by using bacterial
  phenomena-gives precise toxicity status of any
  water body.

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REVIEW ARTICLES

• (1) Quorum Sensing Bacterial phenomenon
• Charu Gera and S. Srivastava (Department of
  Genetics, University of Delhi, South Campus,
  Benito Juarez Road, New Delhi 110 021, India) 
• (2)Quorum sensing and signal interference
  diverse implications
• -Lian-Hui Zhang (Institute of Molecular and Cell
  Biology, 30 Medical
• Drive,Singapore 117609) and Yi-Hu Dong
  (Department of Biological
• Sciences, the National University of Singapore,
  10 Kent Ridge
• Crescent, Singapore 119260).
• (3) Quorum Sensing-How Bacteria Talk to Each
  Other (General Article) -AvantikaLal(3rd year
  student of biochemistry at Sri Venkateswara
• College, Delhi University.)
• (4) Isolation of bioluminescence bacteria its
  application in toxicity detection-A project
  report by Mr. Sachin S. Katti, Mr. Amey M.
  Topkar, Mr. Viral A. Shant.

QUORUM SENSING
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QUORUM SENSING

• Thankyou!!!!!!!