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

Index

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Title
Abstract Biolm- 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

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Future Prospectus of Bioluminescence&Role of quorum sensing in other area Summary Review Articles

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

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

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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. As a result, although several techniques for inhibiting Biofouling by the conventional physical and chemical methods are effective in the initial stage of the biofilm formation, the effect for inhibiting Biofouling after maturation of the biofilm becomes degraded. In order to overcome the problem of conventional methods, new technology has been required to regulate and control characteristics of microorganisms in the reactor, specifically formation and growth of biofilm on the membrane surface. Ramage et al. found that addition of farnesolcan effectively block C. albicans biolm development,leading to the hypothesis that endogenous accumulation offarnesol within biolms may serve as a means for biolm dispersal once a critical cell density is reached. Microarraystudies comparing biolms treated withfarnesol to untreatedcontrols found that the addition of farnesol leads to the decreased expression of hyphae-regulated genes. Ramage et al. also showed that supernatants from maturebiolms inhibited planktonic C. albicans from forming biolms, indicating that farnesol or other supernatant factors mayhelp maintain open spaces or channels within the biolm thatare hypothesized to aid in nutrient inux and waste product efflux by preventing further colonization of the surface. 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. Prior to the bioassay, thestrain was grown overnight on 0.5% yeast extract and 1%tryptone in ltered (0.22-mm) seawater. The inhibitive effects of QS blockers on the production of violacein byC. violaceum cultures were tested according to Martinelliet al. (2004). Briey, the experiments were conducted in 96- well microplates that contained N-hexanoylhomoserinelactones (HHL) at 3.7-10.8M, 100 mLof C. violaceum, andQS blockers. Tested concentrations of QS blockers were103105 M. After exposure for 16 h at 270C, the plateswere dried and the absorbance of each cell well wasmeasured with an automatic plate reader at 590 nm. Cellswithout QS blockers but with HHL and without HHL wereused as the positive and negative controls. In addition, theeffect of QS blockers on bacterial growth was investigated bya comparison of bacterial culture turbidity at 660 nm Result: -All tested compounds signicantly inhibited the productionof violacein by C. violaceum in the presence of HHL (Fig.2). The most effective substanceswere FUR2, which significantly (anova, Dunnettest) inhibited bacterial QS at concentrations of103105M. Violacein formation was reduced by up to90% in comparison with the control, while there was noinhibition of bacterial growth. The results of this experimentsuggest that the tested compounds do indeed interfere withthe signaling system of QS.

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Introduction to Quorum Sensing

A cell-to-cell communication mechanism known as Quorum Sensing (QS) has been found to play a role inP. aeruginosa biolm formation. Quorum sensing (QS) is a way for to exchange information usingcommunication moleculethat bind sensory proteins and thus directly or indirectly affecttranscription and translation. The binding threshold is assumed to be reached oncethe growing population, and hence the concentration of the secreted molecule, attains a certain level. The term communication molecule refers to any Small Molecules (SMs)that is secreted by one organism and changes the behavior of another one. Different systems can be distinguished by the different types of communicationmolecule they use, which are normally also associated with different types of signalsynthesis, import and export, reception and response machinery (Pai and You 2009). The study of cell-to-cell communication and its effects on transcription in unicellularorganisms promises a variety of practical applications. One of them is the possibilityof interfering with intercellular communication systems in pathogenic microbes.

Significance of Quorum-Sensing Systems to Microorganism

Many diverse microorganisms, both Gram-negative and Gram-positive use quorumsensing systems to regulate phenotypes ranging from mating to virulence against the host, antibiotics and production of other metabolites. For example, Streptococcus pneumonia employs a peptide-mediated quorum-sensing system for establishing competence for genetic transformation. In Bacillus subtilis, on the other hand, high cell density contributes to the regulation of at least two different developmental processes development of genetic competence and sporulation. For many years, bacterial cells were considered primarily as selfish individuals, but, in recent years, it has become evident that, they coordinate collective behavior in response to environmental challenges using sophisticated intercellular communication networks. 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. The first organisms in which quorum sensing was observed were MyxobacteriaandStreptomycesspecies. However, 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. When the bacteria are free-living, they are at low concentration and do not luminescent. In the lightproducing organ (photophore) they are highly concentrated (about1011cells/ml) and transcription of luciferase is induced,leading to bioluminescence.
Fig.3: Gene regulation by QS. 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.

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Quorum SensingSignal Molecules

The most important role in QS is played by signaling molecules, called Auto-inducers, which are produced by bacteria. These auto-inducers diffuse through the bacterium membrane into environment. The accumulation of it in the environment takes place if there is a high concentration of bacteria in that space. If this occurs, a special gene will be activated and an increase in signaling molecules production is observed .Therefore the behavior of the bacteria change and the population density will be controlled.

Nature of signal molecule

Microbial-derived signaling molecules can be divided into two main categories (1) Amino Acids and Short Peptide Derivatives, utilized by Gram-positive bacteria and (2) Fatty Acid Derivatives, called Homoserine Lactones (HSLs) utilized by Gram-negative members. Thesignaling mechanism involves subsequent interaction of the signal with intracellular effectors that will induce the pathway for the concerned phenotype. Two general classes of signal molecules, exemplified by Gram-negative and GrampositiveBacteria have been reported. These are: Acyl Homoserine Lactones (AHLs) and Peptide Pheromones respectively. Acyl HSL molecules The family of N-Acyl Homoserine Lactones is almost the universal signal factor in Gram-negative bacteria. The production of AHLs is observed in bacteria, such as P. aeruginosa, Vibrio, Rhizobium producers. AHLs induce the synthesis of compounds interacting with the host organism, such as toxins, antibiotics or exo-enzymes. AHLs have also been found in natural microbial habitats, e.g. biofilm, microbial mats. Although AHL signal molecules from various bacteria are related in structure, they can differ in nature of the Acyl side chain moiety .Depending on the particular auto-inducer, the Acyl group varies from 4 to 14 carbons in length, possesses a hydroxyl group, a carbonyl group, or without a substitution on the third carbon, and is either fully saturated or contains a single carbon carbon doublebond. Many of the individual Acyl HSL species are synthesized by representatives of different bacterial genera. Likewise, many bacterial species can produce more than one type of Acyl HSL. 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. Some peptides interact with membrane bound sensor kinases that transduce a signal across the membrane. Others are transported into the cell by oligopeptide permeases, where they then interact with intracellular receptors. While the Gramnegative bacteria use LuxR-type proteins for auto induction, Gram-positive bacteria use twocomponent adaptive response proteins for the detection of the auto inducers. Thesignaling mechanism is a phosphorylation/ dephosphorylation cascade. The secreted peptide auto inducer
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increases its concentration as a function of the cell population density. Two-component sensor kinases are the detectors for the secreted peptide signals. Phosphorylation of the response regulator activates it, allowing it to bind to the target DNA and alter the transcription of the quorum sensing transcription controlled gene(s). Auto inducer biosynthesis: Homoserine and related compounds are found in most bacteria as intermediates of the MethionineLysineThreoninebiosynthetic pathway.

Measuring Quorum Sensing:

One of the most widely used methods for measuring quorum sensing or, more specifically, bacterial auto inductionis based on the bioluminescent response of V. harveyi, which was developed by Bassleret.al. In this method a cell-free conditioned medium from a culture of interest is incubated with a culture of V. harveyi and the bioluminescent response is recorded. There have been several new techniques intended to more accurately quantify the amount of signal and the level of response exhibited in aquorum system. Frommberger and coworkers describe a liquid chromatography- based concentration and separation method with mass spectrometer determination of various AHLs in bacterial culture. Also, a colorimetric method for determining salicylic acid carboxyl methyltransferase (SAMT) activity has been reported.

Mechanism of Quorum sensing:

The QS mechanism represents one of the most important cell-to cell communication processes. Mechanisms of quorum sensing vary between organisms. General mechanism is as follows. 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). When the inducer binds the receptor, it activates transcription of certain genes, including those for inducer synthesis. There is a low likelihood of a bacterium detecting its own secreted inducer. Thus, in order for gene transcription to be activated, the cell must encounter signaling molecules secreted by other cells in its environment. When only a few other bacteria of the same kind are in the vicinity, diffusion reduces the concentration of the inducer in the surrounding medium to almost zero, so the bacteria produce little inducer. However, as the population grows the concentration of the inducer passes a threshold, causing more inducer to be synthesized. This forms a positive feedback loop, and the receptor becomes fully activated. Activation of the receptor induces the up regulation of other specific genes, causing all of the cells to begin transcription at approximately the same time. This coordinated behavior of bacterial cells can be useful in a variety of situations.

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Bioluminescence- promising application of quorum sensing

Bioluminescence is the most promising application of quorum sensing. Bioluminescent bacteria were used effectively as tool for detecting the presence of toxic chemicals in water source. The following case study was carried out in KIT College of engineering, biotechnology department, Microbiology laboratory, in that the following objectives were achieved1. Isolation of bioluminescent bacteria from natural source: Bioluminescent bacteria Vibrioharveyiwas isolated successfully from the source which is sea water. For isolation of bacteria, the media contended of peptone (5g/l), glycerol (2ml/l), yeast extract (2g/l), agar-agar (15g/l). 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. 0.1 ml of sea water was inoculated by spread plate technique and incubated at 22-250 C for 24 hours. The plate was taken in dark room and light emitting bacterial colonies was observed and isolated. These were sub cultured in same media and characteristics were observed along with Gram nature and motility. This bacterium was cultured successfully in the laboratory both on solid as well as broth media along with retention of its natural bioluminescence. Bacterial Growth pattern was studied correlated with luminescence initiation and termination period. 2. Enhancement in the natural bioluminescence: The natural bioluminescence observed with the techniques like temperature optimization, media optimization and addition of crude auto inducer. These techniques resulted in microorganism with increased bioluminescence as well as better retention time. Optimization of temperature: To study the effect of temperature on the bioluminescence, inoculated cultures were incubated at different temperatures vitz; 250 C, 300 C, 370 C for 18 hours. 50 ml of sea water was inoculated with 2% inoculums. Flasks were observed and flask showing maximum luminescence was noted down. Optimization of media: To obtain maximum level of bioluminescence, medium was optimized. Different concentrations of peptone, yeast extract, glycerol were varied in the media and incubated at 22-250 C for 18 hours were observed. The media composition having peptone 5 g/L, yeast extract 3g/L,&glycerol 3g/L was found to be optimum when the maximum intensity of bioluminescence was noted down. Effect of auto inducer: Autoinducer, 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. In order to enhance the luminescence to
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further level this autoinducer should be provided externally. 200 ml of optimized sea water containing medium was prepared and distributed in two separate flasks. After autoclaving they were inoculated with 2% inoculums and incubated at 22-25 0 C for 18 hrs. After incubation, contents of one flask were transferred aseptically in centrifuge tube and centrifuged at 1000 rpm for 10 min. Supernatant from tubes was taken aseptically in conical flask and kept at RT. Later second flask was divided in two flasks aseptically keeping one as control and other was added with 10 ml medium to be tested, after every 5 min. of incubation.Change in the luminescence was compared after each addition and incubation interval against the control; found that 10 ml of crude autoinducer solution was done luminescence increased after 5 min. of incubation, & after reaching to the threshold value, luminescence started decreasing gradually. 3. Study of effect of toxic chemicals on bioluminescence: Certain representative chemical toxicants like copper sulphate pentahydrate. Zinc sulphate, benzene andtoluene were used in order to observe their effect on bioluminescence. It was proved that as concentration of such chemicals increase, bioluminescence gets decreased. 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. Study on copper sulphate pentahydrateTo study the effect of various concentrations of CuSO4.5H2O on bioluminescence, 100 ml of optimized sea water containing medium was taken in flask and was inoculated with 2% inoculums for 22-250C for 18 hours. Later flask was observed for bioluminescence and was added with 40 ml crude auto inducer and incubated for 10 min. After 10 min increase in luminescence was observed. Later 8 tubes were taken and 5ml broth was added into it. 0.2 % cuso4 5H2O was prepared in D/W and added into tubes as followsBumper Tubes (5 ml.) Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 Vol. of CuSO4.5H2O Amount added added 0 ml. 0.05 ml. 0.10 ml. 0.15 ml. 0.20 ml. 0.25 ml. 0.30 ml. 0.35 ml. 0.40 ml. 1 ml. 0.95 ml. 0.90 ml. 0.85 ml. 0.80 ml. 0.75 ml. 0.70 ml. 0.65 ml. 0.60 ml. of D/W Incubation Time 30 min. 30 min. 30 min. 30 min. 30 min. 30 min. 30 min. 30 min. 30 min.
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All the tubes were kept for incubation for 30 min. The added volume at which luminescence ceased to negligible was noted and effective concentration was calculated. The graph of concentration Vs. cessation of luminescence was plotted and concentration at which luminescence got reduced to its 50% value was obtained graphically. Similarly the effect of chemical toxicants like zinc sulphate, benzene and toluene were studied. The study of toxicant was continued with treating the effluents obtained from pulp industry on bioluminescence bacteria. Reduction in the bioluminescence with increase in the concentration of chemical toxicants was also observed with amazing results.

Future Prospectus of Bioluminescence

Bioluminescence has many promising future aspects. Some of them are: Researchers hope that bioluminescences will be used to track the location of the cells altered with gene therapy. This approach could also illustrate whether these cells were producing the protein after modification. Scientist has discovered how to use make Lucifer in & luciferase in the laboratory. Bye introducing genes into the plants, they hope to be able to identify & control many diseases. Cancer cells & tissue damage from a heart-attack can be detected by injecting Lucifer & luciferase in the suspected disease area.

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. As such, the ability to block or promote these systems provides powerful tools to solve many problems and enhance productivity. Some of the applications are as follows, Staphylococcal subspecies, such as methicillin-resistant S. aureus, remain one of the most opportunistic pathogens found in hospital settings. Quorum sensing phenomena inhibit the development of S. aureus and Staphylococcus epidermidisbiofilms by interfering RNA III Inhibiting Peptide (RIP), a specific inhibitor of quorum sensing with the gene locus agr, which is responsible for staphylococcal toxicity (Balaban and co-workers) Plants have long been known to interact with symbiont bacteria through quorum signaling, and plant pathogens use quorum signaling to colonize their hosts. A review of quorum signaling in plantpathogen symbiont interactions discusses some of the potential
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applications that could arise from these relationships.Part of the biocontrol activity of Bacillus thuringiensisisthrough AHL lactones, an AHL-degrading enzyme. Interestingly, B. thuringiensisdid not inhibit the growth of E. carotovara, but rather,inhibited its virulence and ability to cause soft rot diseasein potatoes. Inhibiting growth in aquatic environments is another potential application of quorum inhibition. In a far different aquatic environment, activated sewage sludge, quorum sensing has been shown to change the characteristics of waste water.

Summary
Far from being singular entities, 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, it shows signs of becoming a promising solution to mans most troublesome problems such as biofilms formation on surgical equipments and other aspects of pathogenicity. Quorum sensing phenomena reveals many interesting facts about the bacterias. For that purpose it is necessary to study in detail the metabolic background of such phenomenon like bioluminescence production. Certain missing links between the process if get cleared, it is possible to use such bacteria toxicity detection in large scale. Also, precise studies of toxicity-bioluminescence relationship, 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, which gives precise toxicity status of any water body. Such kits will involve only microbes, hence easy to use, creates no pollution & most importantly, cost effective.

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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) Quorum Sensing: Bacteria Talk Sense -Costi D. Sifri(Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville). (5) 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.

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