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.

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-

Table 1. List of Seaweeds species collected for the present study SPECIES NAME FAMILY Halimeda gracilis Chlorophyceae Ulva lactuca Chlorophyceae

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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.

Avoid trite clichs What could be worse than the "It wasn't you, it was me" statement? No one believes that, and frankly it's insulting and really makes no sense. Since you aren't breaking up with yourself, how could it not have had something to do with the other person? Avoid using these lines as easy ways out since you know they don't really fool anyone. At least put forth some minimal effort and offer some sort of an explanation if the break-up is sudden.

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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.

In concluding this discussion, the quorum sensing is wider spread among bacterial population then was previously thought, (In Gram positive, Gram negative bacterial communication). Current assays for antimicrobial activities are inadequate because some antibiotic producing bacteria may require the presence of another bacterial species. These findings have important implication for the discovery of novel antimicrobial compounds from marine bacteria and may allow the development of new methods for screening novel compounds active against multidrug resistant bacteria.

NIL NIL NIL NIL Gracillaria edulis 7. GcA+ 8. GcB+ 9. GcC+ 10. GcD+ 11. GcE+ 12. GcF+ GcA- GcB-

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.

Probably not! There is wisdom in knowing yourself: who you are, your beliefs, values, and dreams. And do not pretend to be someone you are not. Fooling another person by making him/her think that you share the same values and beliefs is only going to cause you both disappointments. When you are healthy in all aspects, attractiveness becomes a consequence and not an end. As mentioned in the Klohnen and Luo's research, a person's sense of self-security matters, perhaps even beyond attraction. But remember: do these things for yourself and not for other people. Only then can you truly harness your attractiveness as a person.

Trischman, J.A., D.M.Tapiolas, P.R.Jensen, R.Dwight, W.Fenical, T.C.Mckee, C.M.Ireland, T.J.Stout, J.CIarely. 1994. Salinamide - A and salinamide - B - anti -inflammatory depsipeptides from a marine streptomycete. J.Am.Chem.Soc. 116: 757 -758.

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.

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.

REFERENCES Alim, I., and K. Yuto. 2003. Mc21-A, a Bacteriocidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenotica sp. nov. O-Bc 30T, against Methicillin -Resistant Staphylococcus aureus. Antimicro. Agents Chemoth. 47: 480 - 488.

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.

The above information is supplied of course with the assumption that the relationship is deserving of at least some of the common courtesies that are outlined here. We all know of people who has done something terrible or who isnt worth the effort of striving for an amicable break-up. In either case it's you who will feel better if you handle the situation with as much honesty, dignity and maturity as possible.

CONCLUSION: The present investigation was aimed to study the quorum sensing principle among seaweed epibiotic bacterial organisms. In the past few decades there was no findings of new novel antibacterial class compounds were identified. But, the pathogenic microorganisms show much higher rate of resistant development even to the potential antibiotics. So, there is an urgent need to discover new novel antibiotic compounds. The marine inhabitants such as microorganisms, seaweeds, invertebrates, etc., act as an undepleted source of wide range of natural products among which the seaweeds act as a potential source of antibiotic compounds. Currently the cross species induction / quorum sensing attracts the total attention of researchers in finding new novel drugs against multidrug resistant pathogenic microorganisms. So, the present study aim to find out the capability of seaweed epibiotic bacterial organisms to produce novel drugs against the animal and plant pathogens

RESULTS AND DISCUSSION: QUORUM SENSING/CROSS SPECIES INDUCTION ANALYSIS In the present investigation totally 54 isolates were collected out of seaweed species. Among 54 isolates, 17 of them are producer strain, another 17 are the inducer strain rest of 20 isolates is normal and not showing any signs of activity (Table.2).

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Colony morphology, Gram staining, Motility test, Oxidase test, Catalase test, Indole Production, Methyl red test, Voges Proskauer test, Citrate Utilization test, Triple sugar Iron test, Nitrate reduction test, Lactose fermentation, Urease test

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.

EXPERIMENT NUMBER 2 In this experiment, culture supernatant was obtained as per the procedure given in the experiment 1. 50ml of supernatant was mixed with equal volume of 80% methanol and 1% acetic acid mixture and it was shaked thoroughly in a separating funnel. Finally the methanol and acetic acid fractions were collected and concentrated by evaporation using water bath at 55??C. The viscous colloidal residues were resuspended in 600 microlitre of 50% methanol and it was used for antibacterial assay against different test organism.

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

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

Korber, D. R., J. R.Lawrence, H. M. Lappin-scott, and J. W. Costerton. 1995. Growth of microorganisms on surfaces. In. Microbial. Biofilm, Lapp in - Scott. Cambridge, UK: Cambridge university press.

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

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"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?

So how does attraction figure in relationships? You have probably heard that attraction is a prelude, or a factor towards a relationship. Most probably, at least in the beginning; but attraction alone cannot make a relationship work. It is that attraction that makes you notice a person from the opposite sex, but once you get to know the person more, attraction is just one consideration. Shared values, dreams, and passions become more significant in long-term relationships.

QUORUM SENSING EXPERIMENT NUMBER 1 In this present study, the producer and inducer strains were cross reacted to find out the production of antibiotic compound through quorum sensing. Totally three set of cultures were maintained as follows (along with one as control).

Mearns - Spragg, A., K. G. Boyd, and M. O. Hubble. 1997. Antibiotics from surface associated marine bacteria. Proceedings of the fourth underwater science symposium. The society for underwater Technology, London: 147 - 157.

In culture system B 200ul of 16 hours old producer strain alone was inoculated. In culture system C 200ul of 16 hours old inducer strain alone was inoculated.

MATERIALS AND METHODS: SAMPLE COLLECTION: Seaweed samples were collected from Gulf of Mannar Marine Biosphere Reserve and identified up to species level by using CMFRI bulletin (14) as follows:

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.

Be as Honest as the Day is Long This isn't a trite clich; but rather it's a clich that offers words to live by on a multitude of levels. Lies are insidious in that they must be fed by more even lies in order to be successful. Unless you plan on carrying around a notebook to keep track of all your fibs, white lies and down right untruths, it's best to just live your life honestly, which is after all, the best policy.

Dekievit, T. R., and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 66: 4839-4849. Dimango, E., H. J. Zar, R. Bryan, and A. Prince. 1995. Diverse Pseudomonas aerugniosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin-lnvestig. 96: 2204-2210.

Quorum sensing usually focused on the bacteria growing in homogeneous environment. However few studies have attempted to a study this principle in heterogeneous environment also. In this present investigation we have attempted to study both homogeneous as well as heterogeneous environment. In former one we have isolated producer strain in seaweed eipbionts and it shows inhibitory activity against the inducer organism at the same seaweed epibionts. Later producer strains from seaweed epibionts, were treated with various Vibrio organisms from different environment. The obtain result of this study shows that the producer strain are capable of secreting antibiotic compounds not only to their natural competitors in its own habitate but also to the pathogen inhabiting in a distant related environment.

Evelyn, A., Y. Liming, K.G. Boyd, P. C. Wright, and J. G. Burgess. 2001. The symbiotic role of marine microbes on living surfaces. Hydrobiologia. 461: 37 - 40

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

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.

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

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."

Holmstrom, C, and S. Kjelleberg. 1994. The effect of external biological factors on, settlement of marine invertebrates and new antifouling technologies. Biofouling. 8: 147-160.

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

All the above mentioned biochemical tests were performed by following standard methodology given in the Microbiological Laboratory Manual by James 3.Cappuccino (1999).

U2- U3- U4- U1+/U1- U2+/U2- U3+/U3- U4+/U4- U1- U2- U3- U4- NIL NIL NIL NIL Sediment 17. SA+ SA- SA+/SA- SA- 28

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.

Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199. Msadek, T. 1999. When the going gets tough: survival strategies and environmental signaling network in Bacillus subtilis. Trends Microbiol. 7: 201-207.

c) The normal 20 bacterial strains isolated from 20 algal species were crossed with terrestrial bacteria such as E-coli, Staphylococcus aureus separately

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.

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.

The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing - controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I - generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

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.

Zhang, L., P. J. Murphy, and A. Kerr. 1993. Agrobacterium conjugation and gene regulation by /V-acyl-L-homoserine lactones. Nature 362: 446 - 448.

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.).

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