Interference With Cell-to-cell Signaling: a Potential Therapeutic Approach Against Vibrio Species 

Vijaya baskar .P 1* AND Veera ravi.A 2 1. Department of biotechnology Dr. G.R.D.C.S, Coimbatore. 2. Department of biotechnology, Alagappa University, karaikudi.

a) Among these 17 producers strain 6 strains were isolated from Hypnea musiformis. 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from Sediment.

Is attraction a matter of chemistry? Maybe. According to scientists, the attraction between animals of the opposite sex is all about chemicals called pheromones. The effect of pheromones in behavior of insects is the most studied to date. It has been observed, at least in some experiments, that pheromones are responsible for communication among same species and colony of ants. The horrible odor released by skunks to ward off enemies is said to be a kind of pheromone. Some species of apes rub pheromone-containing urine on the feet of potential mates to attract them.

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Holmstrom, C, D. Rittschof, and S. Kjelleberg. 1992. Inhibition of settlement by larvae of Balanus amphitrite by a surface - colonizing marine bacterium. Appl. Env. Microbiol. 58: 2111 -2115.

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The test organisms Vibrio species were isolated from seaweed as epiphyles, biofilm, sediment and puffer fish by using TCBS medium (Hi media) The pathogenic bacteria were collected from clinical laboratories.

A. Live cells of producer and inducer strains B. Live cells of producer strain alone C. Live cells of inducer strain alone In culture system A 200ul of 16 hours old broth culture of both producer and inducer strains were added to the 15 ml of nutrient broth.

Kell, D. B., A. S. Kaprelyants, and A. Grafen. 1995. Pheromones, Social behaviour and the functions of secondary metabolism in bacteria. Trends in Ecol. and Evol. 10: 126-129.

GcC- GcD- GcE- GcF- Gc A+/GcA- Gc B+/GcB- Gc C+/GcC- Gc D+/GcD- Gc E+ /GcE- Gc F+/GcF- GcA- GcB- GcC-

TEST ORGANISMS: 1. Epiphytic Vibrio from seaweeds 2. Vibrio from primary film 3. Vibrio from Sediments 4. Pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella sp. and Proteus sp

Bainton, N. J., P. Stead, and S.R. Chhabra. 1992. N-(3-oxohexanoyl)-L-homoserine Lactone regulated carbapenem antiobiotic production in Erwinia carotovora. Biochem. J. 288: 997-1004.

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In the present investigation, it was aimed to produce the antibiotics from the seaweed epibionts through quorum sensing principle. The bacterial isolates of seaweed epibionts were identified as species of Pseudomonas and Vibrio from seaweeds Hypnea musiformis and Gracillaria edulis. In this study the Pseudomonas acts as a producer strain and Vibrio as inducer strain. The recent finding says that the seaweed epibionts having potential to control the metabolic activity of competitor organisms. Allison et al., 1998 reported that many bacterial strains up on attaching to a surface produce exopolysaccharides or exopolypeptides. In addition, it has been postulated that exopolysaccharides could mediate the attachment of the bacteria to the surface and induce metabolic changes.

GcA-, GcB-, GcC-, GcD- GcE-, GcF- Gracillaria edulis U1-, U2-, U3-, U4- Ulva lactuca SA- Sediment

In the gram negative bacteria AHSL is an active principle of quorum sensing. Our producer strain is also been identified as Pseudomonas sps. So in these organisms also active principle must falls under the AHSL. The cell-cell signaling mechanism can either require import of the signal and subsequent interaction with intracellular effectors or a two-component signaling system that transducers the information across the membrane. In V. harveyi genetic analysis of the density sensing apparatus has two independent density-sensing systems, and each is composed of a sensor-auto inducer pair; system one is composed of sensor I and Al -1, and system two is composed of sensor 2 and AI-2. The two densities - sensing system are redundant, because a null mutation in either system alone results in expression of hidden genes (Bassler, et al.,1999.).

The results of Vanderivere and Kirchman 1993 suggest that the addition of increased surface by adding sand will induce the exopolymer synthesis through the high cell density dependent system. In the same way the bacterial organisms attached to the surface of seaweed shows alteration of genes expression may be due to the response to the high competitive environment. When cell density increases the competition for space and nutrients are also increased. So the existing bacteria were forced to protect themselves in this competitive environment. Normally in this condition the bacteria will be activated to induce the expression of certain hidden genes in genetic material through quorum sensing. The quorum sensing is principles were active compounds (autoinducer) from bacterial cell will promote the expression of a particular hidden gene of other bacterial organism in a stressed condition.

This experiment does not showed any inhibition zones Bacterial identification The 3 producer and 3 inducer strains which were responded the quorum sensing principles alone were subjected to biochemical analysis for identification. The obtained results revealed that all the producer strains showed sings of Pseudomonas sps and the inducer strains showed signs of Vibrio sps. So, based on the obtained result all the producer strains seems to be a Pseudomonas sps where as all the Inducer strain belongs to the genus vibrio.

Whiteley, M., K.M.Lee, and E.P.Greenberg.1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc.Natl.Acad.Sci.USA.96:13904-13909.

Vandevivere, P., and D. L. Kirchman.1993. Attachment stimulates exopolysaccharide synthesis by a bacterium. Appl. Environ. Microbiol. 59: 3280-3286.

Meains - Spragg, A., M. Bregu, K. G. Boyd, and J. G. Burgess. 1998. Cross species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria. Letters in Applied Microbiology. 27: 142-146.

Lemos, M. L, A. E. Toranzod, and L. J. Barja. 1986. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microbial Ecology. 11: 149-163.

ABSTRACT Bacterial biofilms are sessile communities with high cell density that are ubiquitous in natural, medical, and engineering environments; they are fascinating since they are primitive tissues with an advanced chemical communications network. Currently, there is an explosive amount of biofilm research, most of it with the ultimate aims of biofilm prevention, control, or eradication.The present investigation was aimed to study the cell signaling principle among seaweed epibiotic bacterial organisms , totally 54 bacterial isolates were made out of 20 seaweed species. Among 54 bacterial isolates 17 of them producer, another 17 of them inducer strains rest of 20 isolates were normal that have not showing any signs of activity. The 17 producers and 17 inducer strains were subjected to the cross species induction by quorum sensing principle and found that only 3 of them have responded to the cross species induction in production of antibacterial compound against the respective inducer strains. All the 3 producers and inducer were identified by biochemical analysis and surprisingly that all the 3 producer strain belongs to Genus Pseudomonas and inducer strains were belongs to Genus Vibrio. The supernatant obtain from mixed culture of Pseudomonas & Vibrio shows the antibiotic activity against the various Vibrio species which were isolated from other sources. Such as Vibrio from primary film, Vibrio from sediment Vibrio from seaweeds epibiotics. The obtain results clearly shows the particular Pseudomonas from seaweed epibionts has the capability of producing potential antibiotic compound against wide range of Vibrio species through quorum sensing.

BACTERIAL IDENTIFICATION The organisms responded to the quorum sensing process alone were identified by the following biochemical analysis.

Burgess, J. G., H. Miyashita, H. Sudo, and T. Matsunaga.1991. Antibiotic production by marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: Localization of activity to the chromatophores. FEMS Microbiol. Lett. 84: 301 - 306.

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All the 17 strains were named as PRODUCERS STAINS BrA+, BrB+, BrC+, BrD+, BrE+, BrF+ Hypnea musiformis GcA+, GcB+, GcC+, GcD+, GcE+, GcF+ Gracillaria edulis

Burgess, J. G., M. Elizabeth, M. Bregu, A. Meams-Sprugg, and K. G. Boyd. 1999 Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70. 27-32.

In this present work the totally 54 isolates were screened from 20 different seaweed species out of which 34 species were showed the signs of quorum sensing i.e. 17 producer and 17 inducer strains, but when these organisms where subjected to quorum sensing principle in mixed culture only 3 them have responded. So the present study reveals around 17% of bacterial species isolated from seaweeds and sediment were responded to quorum sensing. According to the results of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% where as that from sea water was only a few percent. In the present study also reveals more or less the same ratio in Pseudomonas spp. was observed. Our results also reveals a results of Kell et al., 1995; Stead et al., 1996; they have said the culture supernatant of Pseudomonas sps known to contain AHLS which induces the production of phenazine antibiotics. In this investigation due to time constraint, It was not attempted to identify the active compound secreted by Pseudomonas through quorum sensing, which may leave the space for the further intensive research in future.

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Allison, D. G., B. Ruiz, C. Sanjose, A. Jaspe, and P.Gilbert. 1998. Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbial Lett. 167: 179-184.

Seaweeds itself secretes secondary metabolites to prevent fouling and grazing. In addition to that epibiotic bacteria on macro algae can also produce antifouling compounds that work in concert with the seaweed derived compounds to protect the seaweed surface. Recent studies have highlighted important roles of epibiotic bacteria colonizing the surface of seaweeds and releasing antifouling compounds. For the past 50 years antibiotics have revolutionized medicine by providing cures for formerly life threatening diseases. However, strains of bacteria have recently emerged that are virtually unresponsive to antibiotics such multidrug resistance, arising mainly through antibiotic misuse, is now recognized as a global health problem. In this situation, it is clear that new classes of antibiotics are urgently needed. Many marine bacteria have been shown to produce secondary metabolites that display antibacterial properties. The first antibiotic from a marine bacterium was identified and characterized in 1966. In addition, bacteria in biofilm on the surface of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environments (Burgess, et al., 1999). Marine epibiotic bacteria, associated with nutrient-rich algal surfaces have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors. Recently a lot new novel antibiotics such as Phenazine, thiomarinol, phenazine-1-carboxylic acid, 1-hydroxyphenazine 2-n-heptylquinol-4-one, 2-n-nonylquinol-4-one pyolipic, loloatins, agrochelin, sesbanimides, pelagiomicins, indomycione and indomycione have been identified from various marine epibiotic bacterial organisms. In particular, some species of the genus Pseudomonas produce both antibiotics and several other bioactive substances. For example, Pseudoalteromonas rubra and Pseudoalteromonas aurantia have been reported to be antibiotic producing bacteria. The phenomenon of higher organisms utilizing their associated microflora for the production of beneficial secondary metabolites is common in the marine environment (Yotsu, et al., 1987). A study of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% whereas that from sea water was only a few percent. In addition, some bacteria that previously did not produce any active compounds have been found to be producing such metabolites when they are exposed to other bacterial species or extra cellular chemical from other bacteria. Bacteria may also produce antimicrobial compounds when they sense the presence of competing organisms. However, few attempts have been made to study such chemical communication between different bacterial species or how this might affect. The secretion of antimicrobial compounds (Mearns-Spragg, et al., 1998). Bacterial communication by the chemical signals for specific function is simply known as Quorum sensing. In which a bacterial population receives input from the environment and elicits an appropriate response (Hiroaki and Kristina. 2003). The term "quorum sensing" describes the ability of a microorganism to perceive and response to diffusible signal molecules. Bacterial cells sense their population density through a sophisticated cell to cell communication system and trigger expression of particular genes. Tne first system of density-dependent regulation was studied in detail with the luminescence of Photobacterium fischeri (formerly known as Vibrio fischeri) by Bassler et al., 1997. Eventually, they discovered that 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamid or N-3-(oxohexanoyl) homoserine lactone (OHHL) was responsible the agent in the broth that induced luminescence. Followed by this many researchers have confirmed that in Gram negative bacteria acyl-homoserine lactone is responsible for the cell to cell communication system.

Bassler, B. L., E. P. Greenberg, and A. M. Sterens. 1997. Cross species induction of luminescence in the quorum - sensing bacterium Vibrio harveyi. J. Bacteriology. 179: 4043-4045.

Mckenney, D., K. E. Brown, and D. G. Allison. 1995. Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication. J. Bacterial 177: 6989 - 6992.

Scientists have been paying close attention to swingers and the development of online swinging communities. They've made extensive research and discovered that swinging can improve relationships, helping people to communicate more freely and be more honest. Not only that people who explored swingers ads and met with some members felt a lot happier, but they declared that their lives are now more exciting and that includes the sexual part. For many couples, engaging in swinging activities with the help of the Internet has been more than they could wish for. The channels of communication were open and they both felt more confident in their sexual nature than before. Online swinging clubs have definitely their merit, especially since they don't judge and tell. They just offer a free-judgment environment for people who are not shy to explore their deepest sexual fantasies and show how far imagination can reach. It is all about sexual attraction in the end and enjoying the very sexuality that we have been gifted with.

Wagner, V. E., D. Bushneil, and L. Passador. 2003. Micro array analysis of Pseudomonas aeruginosa quorum sensing regulons. J. Bacteriol, 185: 2080 - 2095.

Rheinheimer, G.1992.Aquatic microbiology (Wiley, New York), 3rd Ed. Stead, P., B. A. M. Rudd, H. Bradshaw, D. Nobel, and M. J. Dawson. 1996. Induction of phenazine biosynthesis in cultures Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone. FEMS Microbiology Letters 140: 15-22.

All the cultures were incubated at 28??C for 5 days. After the incubation period the cultures were centrifuged at 10,000 rpm for 15mins. The supernatant was collected and subjected to antibacterial assay with respective inducer strain.

Starch hydrolysis test, Protein hydrolysis test, Lipid hydrolysis test, Oxidative / Fermentative test, Salt concentration (0%, 3%, 5%, 7%, 10%), TCBS, Growth in Temperature, 42??C and 47??C

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.

Bassler, B.L.1999. Curr.Opin.Microbiol.2: 582-587.* Bernan, V. S., M. Greenstein, and W. M. Maiese. 1997. Marine microorganisms as a source of new natural products. Adv. Appl. Microbiol. 43: 57 - 89.

Caulerpa racemosa Chlorophyceae Dictyota dichotoma Phaeophyceae Turbinaria ornata Phaeophyceae Padina gymnospora Phaeophyceae Sargassum cinearifolium Phaeophyceae

Burgess, J. G., E. M. Jordan, M. Bregu, and A. Mearns-spragg. 1999. Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70: 27-32.

"Opposites attract" is a law of attraction, at least where electromagnetism is concerned. But are there laws about attraction between two people? "In a world that is full of strangers" as a line in a famous song of the 1980's goes, is there a clear set of rules that allows two people to fall for each other?

Boyd, K.G., A. Mearns -Soragg, G. Briedley, K. Hatzidimitriou, A. Rennie, M. Bregu, M. 0. Hubble, and J. G. Burgess. 1998. Antifouling potential of epiphytic marine bacteria from the surface of marine algae. Plouzane, France: 128 -136.

Does personality figure in sexual attraction? Yes, but so does your perception of a potential mate's personality. According to a research conducted by Klohnen, E.C., & S. Luo in 2003 on interpersonal attraction and personality, a person's sense of self-security and at least the person's perception of his/her partner were found to be strong determinants of attraction in hypothetical situations. What does this tell us? We prefer a certain personality type, which attracts you to a person. But aside from the actual personality of the person, which can only be verified through close interaction through time, it is your perception of your potential partner that attracts you to him/her, whether the person of your affection truly has that kind of personality or not. This could probably account for a statement commonly heard from men and women on their failed relationships: "I thought he/she was this kind of person."

Yotsu, T., Y.Meguro, A. Endo, M. Murate, H. Naold, and T. Yasumoto.1987. Production of tetrodotoxin and its derivatives by Pseudomonas sp. Toxicon 25: 225 - 228

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According to some surveys, about 50% of adult humans can t detect androstenone, unless the concentration is extremely high. Those who can detect androstenone say that its smell is not very pleasant. Androstenone is the main pheromone that men unknowingly secrete that attract women. Both men and women secrete pheromones when they sweat. However, since our body naturally only secretes small amounts...

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