1、Presented By,Miss. Harshada M. SalviB.E.Biotechnology &Mr. Pranav H. Nakhate B.E.Biotechnology,Quorum-sensingThe phenomenon of microbial communication,QUORUM SENSING,Points To Be Discussed,Biolm- a cell-to-cell communication,Bioluminescence- promising application of quorum sensing,Quorum Sensing,QUO
2、RUM SENSING,Biolm- a cell-to-cell communication,Biolm formation .cell-to-cell communication -role in biofilm formation.A Biofilm disadvantageous to humans. Eg. -P. aeruginosa , -C. albicans , -Haemophilus influenzae .,QUORUM SENSING,QUORUM SENSING,Inhibition of Biofilm,Very hard to remove the biofil
3、m , because of its physical & chemical properties.Biofouling of the MBR .Effectivity of farnesol over C. albicans biolm (Ramage et al.)Case study by Martinelli et al. (2004).,QUORUM SENSING,Inhibition of QS by chemical compounds,Used Bacterial mutant strain Chromobacterium violaceum CV026 .Violacein
4、 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.,QUORUM SENSING,Concentration of N-hexanoyl homo serine lactones (HHL) - 3.7-10.8 MIncubation of 270C for 16 hr.No QS Blocke
5、r + HHL= Positive control. No QS Blocker + No HHL = Negative control. Turbidity measurance at 660 nm.,QUORUM SENSING,Result,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 blocke
6、rs interfere with the signalling system.signalling system known as QUORUM SENSING.,QUORUM SENSING,QUORUM SENSING,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 bacte
7、rial 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 biolm formation.,QUORUM SENSING,SO, What actually is QUORUM SENSING.?,QUORUM SENSING,Quorum Sensing :a cell-to-cell
8、communication,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.,QUORUM SENSING,Density de
9、pendent QS,QUORUM SENSING,QS Signal Mechanism,Involves subsequent interaction of the signal with an intracellular effectors.,Cell surface receptors,Signal molecule,Secondary messengers,QUORUM SENSING,Bacteria constantly produce and secrete certain signaling molecules, called as a auto-inducers or ph
10、eromones.Density dependent phenomena.positive feedback loop.,QUORUM SENSING,Q. S. Signal molecules:,Two main categories:-(1) Amino Acids and Short Peptide Derivatives,(2) Fatty Acid Derivatives, called Homoserine Lactones (HSLs).,QUORUM SENSING,Acyl-homoserine lactones (AHLs),Mediate quorum sensing
11、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.,QUORUM SENSING,The peptide pheromone,These are small peptides, regulate gene ex
12、pression in Gram-positive.Interact with membrane bound histidine kinase receptor.,QUORUM SENSING,Bioluminescence- promising application of quorum sensing,Most studied example is V. harveyi .commonly forms symbiotic relationships with some fishes.These marine animals carry a specialized organ called
13、the light organ, in which bacteria like V. harveyi are housed.FMN+NADH +H + FMN REDUCTASE FMNH 2 + NAD + FMNH 2 + RCHO + 0 2 LUCIFERASE FMN + RCOOH + H 2O + LIGHT.,+,QUORUM SENSING,Isolation of bioluminescence bacteria & its application in toxicity detection.,Work has been done in in KIT College of
14、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,QUORUM SENSING,Isolation of bioluminescent bacteria from
15、 environment:-,Media peptone (5g/l), yeast extract (2g/l), agar-agar (15g/l)75 % sea water + 25 % D/WpH - 7Temp.- 220 C-250 C for 24 hoursGram nature and motility,QUORUM SENSING,In Dark,QUORUM SENSING,Enhancement in the natural bioluminescence,QUORUM SENSING,Effect of Autoinducer,200 ml optimized se
16、a 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.,QUORUM SENSING,Study of toxic chemicals,100 ml optimized sea water.,2% innoculum at 22-25 0 C for 18 hrs
17、.,40 ml crude autoinducer( isolated.),10 min. incubation,0.002 gm/ml CuSO4.5H2O.,QUORUM SENSING,C1V1 = C2V2 C1= 0.002 gm/ml.V1=0.30 ml.V2=6 ml.C2 =? Hence, C2=0.1 gm/lit. Which is EC100 value, Hence, EC50 value= 0.05 gm/ lit.,QUORUM SENSING,Applications:-,Gene therapy-to check protein production.Can
18、cer cells , tissue damage detection.Inhibition of biofilm development by RNA III Inhibiting Peptide (RIP).SEWAGE SLUDGE treatment.,QUORUM SENSING,Future Prospectus,LUMINOMETER-an instrument for studying toxicity-bioluminescence relationship.Development of a kit by using bacterial phenomena-gives pre
19、cise toxicity status of any water body.,QUORUM SENSING,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 imp
20、lications-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,Thankyou!,QUORUM SENSING,
