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Quorum-sensing

:
The phenomenon of microbial communication

Presented By, Miss.Harshada M. Salvi
B.E.Biotechnology K.I.T.COEK,Kolhapur E-mail-harshada2503@gmail.com

Mr.Pranav H. Nakhate
B.E.Biotechnology K.I.T.COEK,Kolhapur E-mail-pran.nakhate@gmail.com Mobile no- 09503573593

a cell-to-cell communication Inhibition of Biofilm Introduction to Quorum Sensing Quorum SensingSignal Molecules&Nature of signal molecule Measuring Quorum Sensing Mechanism of Quorum sensing Bioluminescencequorum sensing promising application of Page No 2 3 4 5 6 7 7 8 6 7 8 9 10 11 Future Prospectus of Bioluminescence&Role of quorum sensing in other area Summary Review Articles 10 11 12 Page | 1 .Index Sr. No 1 2 3 4 5 Title Abstract Biofilm.

zinc sulphate. Biofilm. Vibrio harveyi. media & addition of isolated autoinducer for enhancement. benzene and toluene were observed & EC50value was determined graphically.The study was done on biofilm. Bioluminescence. which is a major factor of biofauling inhibited by farnesol. Page | 2 . scientists have discovered that.aureus). Bacteria use these signals to coordinate a wide range of activities.aeruginosa) and pathogenesis (S. which means communication within a bacterial species. which was isolated from sea-water & cultured in laboratory on specific broth with retention of its bioluminescence. called as QuorumSensing. The effect of toxic chemicals like copper sulphate pentahydrate. including bioluminescence (V.. The Bioluminescence phenomenon was studied in detail with marine bacteria. biofilm formation (P. Small Molecules (SM’s). Keywords:Quorum Sensing.Abstract Within the last fifteen years. most groups of bacteria use a rich chemical lexicon to send and receive signals from other bacteria referred to as small molecules (SMs) that bind sensory proteins and thus directly or indirectly affect transcription and translation. fischeri). Attempt had been made to improve original bioluminescence by optimization of temp.

Forexample. albicans biofilm structure and composition canchange under different environmental conditions.Therole of this communication in bacterial biofilm formation is complex and is dependent on the environmental conditions. 1. endo-carditis. cellular differentiation. albicans biofilms that are comprised of abasal yeast layer.a cell-to-cell communication:In nature. Pseudomonas aeruginosa biofilm formed infect humans when they inhale them causing severe on a suture. pneumonia In recent years. Haemophilus influenzae is an important respiratory tract pathogen. A wide variety of extracellular enzymes contribute to the virulence of P. For example anaerobic sulphate reducing bacteria. hemolysins. These include elastase. rhamnolipidbiosurfactants and phospholipase. Biofilm formation is a developmental process in which micro-organisms form multicellular structures with architecture. the micro-organism on which most quorum sensing related studies have been initiated isP.Biofilm. influenzaein patients. protease. Over past decade it is bacteria in the form of biofilm that has been recognized as important causes of various human infections including infections of prosthetic devices. These bacteria can become aerosol and Fig. prostatic and others. though not essential for biofilm formation under many of different conditions. The clinical isolates show that there is a biofilm formation of H. Bacteria. abundant hyphae. dental caries.Biofilm can be host for the pathogenic Legionella. It is the important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. Furthermore. aeruginosa.and biocide resistance . Page | 3 . enhanced resistance to stressesand. Also the so-called iron-oxidizing bacteria can cause corrosion of metal. and a calcofluor-bindingextracellular matrix. altered gene expression patterns. in P. aeruginosa is an important human pathogen which is responsible for opportunistic infections in cancer. it has been shown thatthe formation of hyphae is important. exotoxin A. AIDS and cystic fibrosis (CF) patients.aeruginosa. resulting in expensive repairs of leaking pipes. in some cases. A cell-to-cell communication has been found to play an important role in biofilm formation. AcylHomoserine Lactones(AHL) signals are important for a number of biofilmproperties.While C. the bulk of bacterial biomass is believed to exist as an adherent community of cells called a Biofilm.P. These bacteria produce sulphuric acid which can cause corrosion of metal pipes. aeruginosabiofilms. including the formation channels. pneumonia in cystic fibrosis. The bacterium causes acute otitis. A Biofilm can contain several bacteria that can produce corrosive chemicals. Several reports describe C. cell dispersal. These exo-factors are collectively capable of causing extensive tissue damage in humans and other mammals.

(2004).7-10.8M. which significantly (anova. violaceum in the presence of HHL (Fig. 100 mLof C. new technology has been required to regulate and control characteristics of microorganisms in the reactor.leading to the hypothesis that endogenous accumulation offarnesol within biofilms may serve as a means for biofilm dispersal once a critical cell density is reached. Ramage et al.2). The most effective substanceswere FUR2. andQS blockers.Inhibition of Biofilm It is not easy to remove the biofilm which is a major factor of Biofouling of the MBR because the biofilm has high resistance even in external physical and chemical shocks. the effect for inhibiting Biofouling after maturation of the biofilm becomes degraded. Violacein formation was reduced by up to90% in comparison with the control. albicans from forming biofilms. the experiments were conducted in 96. Inhibition of QS by chemical compounds The bacterium Chromobacterium violaceum CV026 (a mini-Tn5 mutant) was used as an indicator organism for QS byquantifying violacein synthesis. also showed that supernatants from maturebiofilms inhibited planktonic C. The inhibitive effects of QS blockers on the production of violacein byC. theeffect of QS blockers on bacterial growth was investigated bya comparison of bacterial culture turbidity at 660 nm Result: -All tested compounds significantly inhibited the productionof violacein by C. violaceum cultures were tested according to Martinelliet al. Tested concentrations of QS blockers were103–105 M. thestrain was grown overnight on 0. Microarraystudies comparing biofilms treated withfarnesol to untreatedcontrols found that the addition of farnesol leads to the decreased expression of hyphae-regulated genes. while there was noinhibition of bacterial growth. albicans biofilm development. the plateswere dried and the absorbance of each cell well wasmeasured with an automatic plate reader at 590 nm. Dunnettest) inhibited bacterial QS at concentrations of103–105M. found that addition of farnesolcan effectively block C. After exposure for 16 h at 270C. Prior to the bioassay. specifically formation and growth of biofilm on the membrane surface. In addition. violaceum. In order to overcome the problem of conventional methods.5% yeast extract and 1%tryptone in filtered (0. The results of this experimentsuggest that the tested compounds do indeed interfere withthe signaling system of QS. Ramage et al.22-mm) seawater. Cellswithout QS blockers but with HHL and without HHL wereused as the positive and negative controls. Page | 4 . although several techniques for inhibiting Biofouling by the conventional physical and chemical methods are effective in the initial stage of the biofilm formation. As a result. indicating that farnesol or other supernatant factors mayhelp maintain open spaces or channels within the biofilm thatare hypothesized to aid in nutrient influx and waste product efflux by preventing further colonization of the surface. Briefly.well microplates that contained N-hexanoylhomoserinelactones (HHL) at 3.

One of them is the possibilityof interfering with intercellular communication systems in pathogenic microbes. In Bacillus subtilis. on the other hand. Quorum sensing (QS) is a way for to exchange information using‘communication molecule’that bind sensory proteins and thus directly or indirectly affecttranscription and translation. aeruginosa biofilm formation. bacterial cells were considered primarily as selfish individuals.Introduction to Quorum Sensing A cell-to-cell communication mechanism known as Quorum Sensing (QS) has been found to play a role inP. and hence the concentration of the secreted molecule. they coordinate collective behavior in response to environmental challenges using sophisticated intercellular communication networks.leading to bioluminescence. they are at low concentration and do not luminescent. The study of cell-to-cell communication and its effects on transcription in unicellularorganisms promises a variety of practical applications.3: Gene regulation by QS. antibiotics and production of other metabolites. QS Molecule (represented by black dots) is produced throughout the microbial growth and induces the transcription of target genes only upon reaching a threshold concentration. The binding threshold is assumed to be reached oncethe growing population. attains a certain level. in recent years. For example. reception and response machinery (Pai and You 2009). When the bacteria are free-living. Cell-to-cell communication between bacteria is mediated by small diffusible signal molecules that trigger changes in gene expression in response to fluctuations in population density. Fig. Significance of Quorum-Sensing Systems to Microorganism Many diverse microorganisms. it has become evident that. The first organisms in which quorum sensing was observed were MyxobacteriaandStreptomycesspecies. Streptococcus pneumonia employs a peptide-mediated quorum-sensing system for establishing competence for genetic transformation. but. In the lightproducing organ (photophore) they are highly concentrated (about1011cells/ml) and transcription of luciferase is induced. Page | 5 . which are normally also associated with different types of signalsynthesis. the most popular example is the regulation of light production in Vibrio fisheri. abioluminescent bacterium that lives symbiotic in the light-producing organ of deep-sea bobtail squid. Different systems can be distinguished by the different types of communicationmolecule they use. high cell density contributes to the regulation of at least two different developmental processes – development of genetic competence and sporulation. However. The term ‘communication molecule’ refers to any Small Molecules (SM’s)that is secreted by one organism and changes the behavior of another one. For many years. both Gram-negative and Gram-positive use quorumsensing systems to regulate phenotypes ranging from mating to virulence against the host. import and export.

exemplified by Gram-negative and GrampositiveBacteria have been reported. Gram-positive bacteria use twocomponent adaptive response proteins for the detection of the auto inducers.  The peptide pheromone Gram-positive quorum-sensing systems typically make use of small posttranscriptionally processed peptide signal molecules these peptides are usually secreted by ATPbinding cassette (ABC) transporters. These auto-inducers diffuse through the bacterium membrane into environment. microbial mats. The secreted peptide auto inducer Page | 6 . While the Gramnegative bacteria use LuxR-type proteins for auto induction. utilized by Gram-positive bacteria and (2) Fatty Acid Derivatives. such as P. AHLs induce the synthesis of compounds interacting with the host organism.Depending on the particular auto-inducer. Two general classes of signal molecules. Thesignaling mechanism involves subsequent interaction of the signal with intracellular effectors that will induce the pathway for the concerned phenotype. e. called Homoserine Lactones (HSL’s) utilized by Gram-negative members. Although AHL signal molecules from various bacteria are related in structure. the Acyl group varies from 4 to 14 carbons in length.g. Rhizobium producers. Some peptides interact with membrane bound sensor kinases that transduce a signal across the membrane. Likewise. called Auto-inducers. a carbonyl group. biofilm. Thesignaling mechanism is a phosphorylation/ dephosphorylation cascade. antibiotics or exo-enzymes. aeruginosa.Therefore the behavior of the bacteria change and the population density will be controlled. The production of AHLs is observed in bacteria. Vibrio. The accumulation of it in the environment takes place if there is a high concentration of bacteria in that space. they can differ in nature of the Acyl side chain moiety . and is either fully saturated or contains a single carbon– carbon doublebond. possesses a hydroxyl group. which are produced by bacteria. AHLs have also been found in natural microbial habitats. If this occurs.  Acyl HSL molecules The family of N-Acyl Homoserine Lactones is almost the universal signal factor in Gram-negative bacteria. such as toxins. or without a substitution on the third carbon. many bacterial species can produce more than one type of Acyl HSL. Many of the individual Acyl HSL species are synthesized by representatives of different bacterial genera. where they then interact with intracellular receptors. Nature of signal molecule Microbial-derived signaling molecules can be divided into two main categories (1) Amino Acids and Short Peptide Derivatives. Others are transported into the cell by oligopeptide permeases. a special gene will be activated and an increase in signaling molecules production is observed .Quorum SensingSignal Molecules The most important role in QS is played by signaling molecules. These are: Acyl Homoserine Lactones (AHLs) and Peptide Pheromones respectively.

Activation of the receptor induces the up regulation of other specific genes. Two-component sensor kinases are the detectors for the secreted peptide signals. Frommberger and coworkers describe a liquid chromatography. Also. There is a low likelihood of a bacterium detecting its own secreted inducer.al. However. harveyi and the bioluminescent response is recorded. the cell must encounter signaling molecules secreted by other cells in its environment. General mechanism is as follows. more specifically. In this method a cell-free conditioned medium from a culture of interest is incubated with a culture of V.based concentration and separation method with mass spectrometer determination of various AHLs in bacterial culture. causing more inducer to be synthesized. including those for inducer synthesis. This forms a positive feedback loop. in order for gene transcription to be activated. Thus. harveyi. Page | 7 . bacterial auto inductionis based on the bioluminescent response of V. a colorimetric method for determining salicylic acid carboxyl methyltransferase (SAMT) activity has been reported. and the receptor becomes fully activated. When only a few other bacteria of the same kind are in the vicinity. causing all of the cells to begin transcription at approximately the same time. Measuring Quorum Sensing: One of the most widely used methods for measuring quorum sensing or. it activates transcription of certain genes. which was developed by Bassleret. as the population grows the concentration of the inducer passes a threshold. Bacteria that use quorum sensing constantly produce and secrete certain signaling molecules (called auto inducers or pheromones). These bacteria also have a receptor that can specifically detect the signaling molecule (inducer). so the bacteria produce little inducer. There have been several new techniques intended to more accurately quantify the amount of signal and the level of response exhibited in aquorum system.  Auto inducer biosynthesis: Homoserine and related compounds are found in most bacteria as intermediates of the Methionine–Lysine–Threoninebiosynthetic pathway. Mechanisms of quorum sensing vary between organisms. When the inducer binds the receptor. allowing it to bind to the target DNA and alter the transcription of the quorum sensing transcription controlled gene(s). Mechanism of Quorum sensing: The QS mechanism represents one of the most important cell-to cell communication processes.increases its concentration as a function of the cell population density. diffusion reduces the concentration of the inducer in the surrounding medium to almost zero. This coordinated behavior of bacterial cells can be useful in a variety of situations. Phosphorylation of the response regulator activates it.

The plate was taken in dark room and light emitting bacterial colonies was observed and isolated. All these components were dissolved in combination of (75% sea water +25% distilled water) and pH was adjusted to 7 and allowed to solidify in Petri plate. glycerol were varied in the media and incubated at 22-250 C for 18 hours were observed. Bioluminescent bacteria were used effectively as tool for detecting the presence of toxic chemicals in water source.1 ml of sea water was inoculated by spread plate technique and incubated at 22-250 C for 24 hours.promising application of quorum sensing Bioluminescence is the most promising application of quorum sensing. Isolation of bioluminescent bacteria from natural source: Bioluminescent bacteria Vibrioharveyiwas isolated successfully from the source which is sea water. In order to enhance the luminescence to Page | 8 . the quorum sensing regulatory signaling agent is secreted by the bioluminescent bacteria in surrounding media which when reaches a particular threshold concentration enhances the bioluminescence. Bacterial Growth pattern was studied correlated with luminescence initiation and termination period. Enhancement in the natural bioluminescence: The natural bioluminescence observed with the techniques like temperature optimization. yeast extract 3g/L. This bacterium was cultured successfully in the laboratory both on solid as well as broth media along with retention of its natural bioluminescence. 0. For isolation of bacteria. yeast extract (2g/l). the media contended of peptone (5g/l). Flasks were observed and flask showing maximum luminescence was noted down. Microbiology laboratory. The media composition having peptone 5 g/L.  Optimization of media: To obtain maximum level of bioluminescence.Bioluminescence. medium was optimized. These were sub cultured in same media and characteristics were observed along with Gram nature and motility. agar-agar (15g/l). These techniques resulted in microorganism with increased bioluminescence as well as better retention time. 300 C. 50 ml of sea water was inoculated with 2% inoculums. biotechnology department. in that the following objectives were achieved1.  Effect of auto inducer: Autoinducer.&glycerol 3g/L was found to be optimum when the maximum intensity of bioluminescence was noted down.  Optimization of temperature: To study the effect of temperature on the bioluminescence. 250 C. yeast extract. glycerol (2ml/l). 2. Different concentrations of peptone. media optimization and addition of crude auto inducer. inoculated cultures were incubated at different temperatures vitz. 370 C for 18 hours. The following case study was carried out in KIT College of engineering.

after every 5 min. 0.15 ml. 30 min. 100 ml of optimized sea water containing medium was taken in flask and was inoculated with 2% inoculums for 22-250C for 18 hours.20 ml. 30 min.40 ml.30 ml. luminescence started decreasing gradually. It was proved that as concentration of such chemicals increase.further level this autoinducer should be provided externally. found that 10 ml of crude autoinducer solution was done luminescence increased after 5 min. Zinc sulphate.Change in the luminescence was compared after each addition and incubation interval against the control.60 ml.90 ml. 0. 0. Page | 9 . 0. Later second flask was divided in two flasks aseptically keeping one as control and other was added with 10 ml medium to be tested. 0.2 % cuso4 5H2O was prepared in D/W and added into tubes as followsBumper Tubes (5 ml. of D/W Incubation Time 30 min. 1 ml. Later flask was observed for bioluminescence and was added with 40 ml crude auto inducer and incubated for 10 min. 30 min. 0. of CuSO4. 0. 3. bioluminescence gets decreased.5H2O Amount added added 0 ml. 200 ml of optimized sea water containing medium was prepared and distributed in two separate flasks.65 ml. 0. 0.75 ml. 0.70 ml. of incubation. 0. 30 min.25 ml. 0. 0. After autoclaving they were inoculated with 2% inoculums and incubated at 22-25 0 C for 18 hrs.95 ml. 30 min.85 ml.05 ml.10 ml. 0. Also study on the effluent obtained from paper and pulp industry showed that based on EC 50 values portability of any water sample can be decided by this method. Supernatant from tubes was taken aseptically in conical flask and kept at RT.5H2O on bioluminescence. 0.35 ml. 30 min. benzene andtoluene were used in order to observe their effect on bioluminescence. After 10 min increase in luminescence was observed.) Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 Vol. 0. & after reaching to the threshold value. Later 8 tubes were taken and 5ml broth was added into it. Study of effect of toxic chemicals on bioluminescence: Certain representative chemical toxicants like copper sulphate pentahydrate. After incubation. contents of one flask were transferred aseptically in centrifuge tube and centrifuged at 1000 rpm for 10 min. 0.80 ml. 30 min. 30 min. of incubation.  Study on copper sulphate pentahydrateTo study the effect of various concentrations of CuSO4.

 Scientist has discovered how to use make Lucifer in & luciferase in the laboratory. As such. aureus. A review of quorum signaling in plant–pathogen symbiont interactions discusses some of the potential Page | 10 . and plant pathogens use quorum signaling to colonize their hosts. Some of them are: Researchers hope that bioluminescence’s will be used to track the location of the cells altered with gene therapy. The study of toxicant was continued with treating the effluents obtained from pulp industry on bioluminescence bacteria. they hope to be able to identify & control many diseases. Some of the applications are as follows. Quorum sensing phenomena inhibit the development of S. Future Prospectus of Bioluminescence Bioluminescence has many promising future aspects. benzene and toluene were studied. Reduction in the bioluminescence with increase in the concentration of chemical toxicants was also observed with amazing results.All the tubes were kept for incubation for 30 min. a specific inhibitor of quorum sensing with the gene locus agr. Similarly the effect of chemical toxicants like zinc sulphate. which is responsible for staphylococcal toxicity (Balaban and co-workers)  Plants have long been known to interact with symbiont bacteria through quorum signaling. cessation of luminescence was plotted and concentration at which luminescence got reduced to its 50% value was obtained graphically.  Cancer cells & tissue damage from a heart-attack can be detected by injecting Lucifer & luciferase in the suspected disease area. such as methicillin-resistant S. Role of quorum sensing in other area A great number of bacteria employ quorum-sensing for regulation of various phenotypes as a part of their pathogenic or symbiotic lifestyles. The graph of concentration Vs. Bye introducing genes into the plants. The added volume at which luminescence ceased to negligible was noted and effective concentration was calculated.  Staphylococcal subspecies. remain one of the most opportunistic pathogens found in hospital settings. This approach could also illustrate whether these cells were producing the protein after modification. the ability to block or promote these systems provides powerful tools to solve many problems and enhance productivity. aureus and Staphylococcus epidermidisbiofilms by interfering RNA III Inhibiting Peptide (RIP).

quorum sensing has been shown to change the characteristics of waste water.  Inhibiting growth in aquatic environments is another potential application of quorum inhibition. activated sewage sludge. creates no pollution & most importantly. thuringiensisdid not inhibit the growth of E. Certain missing links between the process if get cleared. B. hence easy to use. it is now apparent that bacteria exist in multifaceted communities and are constantly communicating with each other. The importance of this field of study is. cost effective. Also. precise studies of toxicity-bioluminescence relationship. For that purpose it is necessary to study in detail the metabolic background of such phenomenon like bioluminescence production. Interestingly.Part of the biocontrol activity of Bacillus thuringiensisisthrough AHL lactones. Quorum sensing phenomena reveals many interesting facts about the bacteria’s. but rather. which gives precise toxicity status of any water body. sophisticated instruments like luminometer are required which are capable of measuring exact light intensities emitted by bacteria. It is also possible to develop a kit by using these bacterial phenomena. Summary Far from being singular entities.inhibited its virulence and ability to cause soft rot diseasein potatoes. carotovara. Page | 11 .applications that could arise from these relationships. it is possible to use such bacteria toxicity detection in large scale. Such kits will involve only microbes. it shows signs of becoming a promising solution to man’s most troublesome problems such as biofilms formation on surgical equipments and other aspects of pathogenicity. In a far different aquatic environment. an AHL-degrading enzyme.

Charlottesville). the National University of Singapore. University of Delhi. Singapore 119260). Benito Juarez Road. Srivastava (Department of Genetics.) (4) Quorum Sensing: Bacteria Talk Sense -Costi D. University of Virginia Health System. 30 Medical Drive. New Delhi 110 021. Mr. Sifri(Division of Infectious Diseases and International Health. Katti. Topkar. Page | 12 . (3) Quorum Sensing-How Bacteria Talk to Each Other (General Article) -AvantikaLal(3rd year student of biochemistry at Sri Venkateswara College. Delhi University. 10 Kent Ridge Crescent.REVIEW ARTICLES (1) Quorum Sensing: Bacterial phenomenon Charu Gera and S. Shant. Mr. Viral A.Singapore 117609) and Yi-Hu Dong (Department of Biological Sciences. Sachin S. Amey M. India) (2) Quorum sensing and signal interference: diverse implications -Lian-Hui Zhang (Institute of Molecular and Cell Biology. (5) Isolation of bioluminescence bacteria &its application in toxicity detection A project report by Mr. South Campus.