University of Cambridge

Studies on Quorum Sensing Principle Among Seaweed Epibiotic Bacterial Organisms

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 -ortalities 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). 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.
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.

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:

Table 1. List of Seaweeds species collected for the present study

SPECIES NAME FAMILY
Halimeda gracilis Chlorophyceae
Ulva lactuca Chlorophyceae
Microdictyon tenunis Chlorophyceae
Chondrococcus hornemonii Chlorophyceae
Enteromorpha intestinalis Chlorophyceae
Caulerpa cupressoides Chlorophyceae
Caulerpa racemosa Chlorophyceae
Dictyota dichotoma Phaeophyceae
Turbinaria ornata Phaeophyceae
Padina gymnospora Phaeophyceae
Sargassum cinearifolium Phaeophyceae
Dictyota batryensis Phaeophyceae
Sargassum sps Phaeophyceae
Hypnea musciformis Rhodophyceae
Acanthophora dendroides Rhodophyceae
Jania rubens Rhodophyceae
Hypnea valentiae Rhodophyceae
Hypnea pannose Rhodophyceae
Hypnea esperi Rhodophyceae
Acanthophora spicifera Rhodophyceae

ISOLATION OF EPIPHYTIC BACTERIA
The collected seaweed samples were thoroughly washed with sterile seawater to removes the loosely attached bacteria/particles. Seaweed fronds were scrubbed with sterile cotton swabs to obtain epiphytic bacteria. Epiphytic bacterial organism in the swab were inoculated in sterile peptone broth (50% sea water) and incubated at 28°C in an incubated shaker (220 rpm / min) for overnight. After the incubation period the enriched cultures were serially diluted up to 10-8 concentration and 200 microlitre of each diluted samples were transferred into the nutrient agar plate (50% sea water). The plates were incubated at 28°C for 5 days and the plates with crowded colonies were selected. In the crowded plates those colonies, which showed the sign of inhibition zone around its margin to the neighboring colony, were selected and considered as producer strain. The neighboring sensitive colonies were treated as inducer strain. Both producer and inducer strains were streaked repeatedly until to get pure culture. The pure culture were properly labeled and subjected to the quorum sensing analysis.

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).

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.

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.

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.

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.

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

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.

ANTIBIOTIC ASSAYS
Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37°C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

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

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
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

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

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).

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.

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
U1+, U2+, U3+, U4+ Ulva lactuca
SA+ Sediment

INDUCER STRAIN
BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis
GcA-, GcB-, GcC-, GcD- GcE-, GcF- Gracillaria edulis
U1-, U2-, U3-, U4- Ulva lactuca
SA- Sediment

In this experiment among 17 Producer and Inducer strains only 3 of them have responded to the quorum sensing principle. (BrB+/Bo-?, (GcC+/GcC) and (SA+/SA-)
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)
Hypnea musiformis 1. BrA+2. BrB+3. BrC+4. BrD+5. BrE+6. BrF+ BrA-BrB-BrC-BrD-BrE-BrF- Br A+ /Br A-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- NIL39NILNILNILNIL
Gracillariaedulis 7. GcA+8. GcB+9. GcC+10. GcD+11. GcE+12. GcF+ GcA-GcB-GcC-GcD-GcE-GcF- Gc A+/Gc-Gc B+/Gc-Gc C+/Gc-Gc D+/Gc-Gc E+ /GcE-Gc F+/Gc- GcA-GcB-GcC-GcD-GcE-GcF- NILNIL26NILNILNIL
Ulva lactuca 13. U1+14: U2+15. U3+16. U4+ U1-U2-U3-U4- U1+/U0-U2+/U0-U3+/U0-U4+/U4- U1-U2-U3-U4- NILNILNILNIL
Sediment 17. SA+ SA- SA+/SA- SA- 28

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

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.

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.

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.

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.

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 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].

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.

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.

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.

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