10/16/2019

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.


*Corresponding author e-mail: vijay10bas@yahoo.co.in INTRODUCTION We are living in a microbial planet. About 71 % of the surface of this planet is covered by sea water. A typical milliliter of seawater contains 103 fungal cells, 106 bacteria, and 107 viruses, including pathogens that cause widespread mortalities and microbes that initiate fouling of host surfaces (Rheinheimer, 1992). Thus, marine plants and animals are continually exposed to high concentrations of potentially harmful microbes. These microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, 'reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001).
 




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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.

Table 2: The results of Quorum Sensing analysis of epibiotic bacterial isolates from seaweeds. Seaweed sample Producer organism Inducer organism Cross-species producer with inducer Cross-species supernatant test with inducer Zone of clearance (mm)

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

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].

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.

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).

Hiroaki, S., and K. M. Smith. 2003. Molecular mechanisms of bacterial quorum sensing as a new drug target. Curr. Opin. Chem. Biol. 7: 586-591. Hoang, T., Y. Ma, R. J. Stern, M. R. Meneil, and H. P. Schwezer. 1999. Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of Mycobacterium tuberculosis RmID and Pseudomonas aeruginosa Lasl and Rhll proteins, and functional analysis of purified Rhll. Gene. 237: 361 - 371.

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.

Br B+ /Br B- Br C+/Br C- Br D+/Br D- Br E+/Br E- Br F+/Br F- BrA- Br B- BrC- BrD- BrE- BrF- NIL 39

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-

GcD- GcE- GcF- NIL NIL 26 NIL NIL NIL Ulva lactuca 13. U1+ 14: U2+ 15. U3+ 16. U4+ U1-

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

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

Liming, Y., G. K. Boyd, and J. G. Burgess. 2002. Surface induced attachment induced production of Antimicrobial compounds by marine epibiotic Bacteria using modified Roller Bottle. Mar. Biotechnology. 4: 356 - 366.

Br B+ /Br B- Br C+/Br C- Br D+/Br D- Br E+/Br E- Br F+/Br F- BrA- Br B- BrC- BrD- BrE- BrF- NIL 39

There have been numerous studies conducted with couples and even twins put in the same position at bars as a social experiment. The overwhelming outcome of the studies was that the people who applied human pheromones were approached more often, and the couples hugged, kissed and had sex more often, and the twin that applied the pheromones received double the attention and people approaching as the twin that did not apply them.

Though not proven highly effective at alluring the opposite sex as some products claim, experiments have shown that pheromones do affect human behavior as well. There is evidence that females may synchronize their menstrual cycles based on the presence of pheromones in other females sweat.

So yes, pheromones do work, and they can be an excellent way to attract more attention from men and women alike, or even to do your own little "experiment". Why not?

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.

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.

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

We suggest that the above said mechanisms in Pseudomonas with quorum sensing principle might have occurred in the present study also. This induces the bacteria Pseudomonas in epibiotic seaweeds to secrete certain active compound against to the competitor Vibrio species.

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.

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].

In gram positive bacteria peptide and derivative peptide based signaling molecules seem to be the predominant mode of communication. During high cell density the marine bacteria can produce enzymes, surfactants, toxins, and antibiotics by the chemical signal communication. Marine epibiotic bacteria are also known to produce compounds active against drug resistant hospital pathogen by the cross species induction method. Building on assays described by Austin (Billaud and Austin 1990) a screening procedure has been developed in which marine bacteria are challenged by exposing them to terrestrial bacteria prior to assay of antimicrobial compounds. Hence in this present investigation it is proposed to find out the ability of sea weed epibiotic bacterial organism to produce antibacterial compounds through quorum sensing.

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.

Writing this composition on colognes that contain pheromones was a significant contribution of ours in the world of literature. Make this contribution worthwhile by using it.



Icebreaker Pheromone Cologne



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.

Hiroaki, S., and K. M. Smith. 2003. Molecular mechanisms of bacterial quorum sensing as a new drug target. Curr. Opin. Chem. Biol. 7: 586-591. Hoang, T., Y. Ma, R. J. Stern, M. R. Meneil, and H. P. Schwezer. 1999. Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of Mycobacterium tuberculosis RmID and Pseudomonas aeruginosa Lasl and Rhll proteins, and functional analysis of purified Rhll. Gene. 237: 361 - 371.

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.

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.

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-

Cappuccino J. G., and N. Shermann. 1999. Microbiology, A Laboratory manual, Fourth edition, Addison Wesley, New York. Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of cell to cell signals in the development of a bacterial biofilm. Science. 280: 295-298.

The problem is, Pheromone production varies from subject to subject. While some may possess an abundance of this, well, hormone of desire, others seem to suffer from a shortage of the same.

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.

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

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.

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.

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.

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

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).

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.

Hypnea musiformis 1. BrA+ 2. BrB+ 3. BrC+ 4. BrD+ 5. BrE+ 6. BrF+ BrA- BrB- BrC- BrD- BrE- BrF- Br A+ /Br A-

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

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:

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.

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.

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


Make sure that your other actions do not impede the work of pheromones
Since the pheromones work on a subconscious level without the male even knowing it, it is a greater attracting force than your actions. It is a magnet working for you and if you know your man better you will be able to add to that bond of attraction, you have everything working for you and no negative factors. Hence pheromones work for you.



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Author Dave Thomson
 
 
 


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