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Keywords:

  • brown alga;
  • Colpomenia sinuosa;
  • epibiotic bacteria;
  • quorum-sensing inhibitor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Aims:  Several Gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing (QS) signals to regulate various biological functions. Similarly, various bacteria can stimulate, inhibit or inactivate QS signals in other bacteria by producing molecules called as quorum-sensing inhibitors (QSI). Our aim was to screen and identify the epibiotic bacteria associated with brown algae for their ability of producing QS-inhibiting activity.

Methods and Results:  QSI screenings were conducted on several epibiotic bacteria isolated from a marine brown alga Colpomenia sinuosa, using Serratia rubidaea JCM 14263 as an indicator organism. Strain JCM 14263 controls the production of red pigment, prodigiosin by AHL QS. Out of 96 bacteria, which were isolated from the surface of the brown alga, 12% of strains showed the ability to produce QSI, which was observed from the pigmentation inhibition on Ser. rubidaea JCM 14263 without affecting its growth. Phylogenetic analysis using 16S rRNA gene sequencing method demonstrated bacterial isolates showing QS inhibition-producing bacteria belonging to the Bacillaceae (Firmicutes), Pseudomonadaceae (Proteobacteria), Pseudoalteromonadaceae (Proteobacteria) and Vibrionaceae (Proteobacteria).

Conclusion:  An appreciable percentage of bacteria isolated from the brown alga produced QSI-like compounds.

Significance and Impact of the Study:  The screening method using Ser. rubidaea described in this report will facilitate the rapid identification of QSI-producing bacteria from marine environment. This study reveals new avenue for future environmental applications. This study also suggests that these algal epibiotic bacteria may play a role in the defensive mechanism for their host by producing QSI or QSI-like compounds to suppress the settlement of other competitive bacteria.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Quorum sensing (QS) is a process of intercellular communication in which bacteria secrete small and membrane-diffusible signal molecules called as autoinducers. In Gram-negative bacteria, N-acyl homoserine lactone (AHL)-mediated cell–cell signalling plays a role in regulating bacterial functions such as the biosynthesis of antibiotics (Pierson et al. 1994), production of virulence factors, exopolysaccharide biosynthesis (Cha et al. 1998), pigment production, bioluminescence as well as biofilm formation (McClean et al. 1997). In Gram-positive bacteria, cell–cell signalling is mediated not by AHLs, but rather by γ-butyrolactones and post-translationally modified peptides (Brelles-Marino and Bedmar 2001).

Recently, there is growing interest in discovering ways to disrupt or block QS-signalling in bacteria. It is proposed that various bacteria can stimulate, inhibit or inactivate QS signals in other bacteria by producing molecules called as quorum-sensing inhibitors (QSI) or QS antagonists, which are AHL-like molecules that can bind to QS response regulators, but fail to activate them. QSI compounds would have potential biotechnological applications in the near future, because blocking cell–cell communication within or among the bacterial species could prevent the spread of pathogenicity, formation of biofilm, etc. An earlier study has shown that AHL signal molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that formation of biofilm can be controlled by interfering with the bacterial cell–cell communication (Davies et al. 1998). Hence, it is plausible that QSI may represent a natural, wide spread and antimicrobial strategy utilized by bacteria with significant impact on biofilm formation (Bauer and Robinson 2002).

Many sessile marine invertebrates such as sponges and macroalgae have evolved defence mechanisms against fouling by producing metabolites that can inhibit the settlement and growth of other organism. However, some macroalgae which are lacking chemical and nonchemical defences are believed to rely on the surface-associated bacteria. Colpomenia sinuosa belongs to the order Phaeophyceae and is commonly called as brown bag weed. We observed that specimens of brown alga C. sinuosa, found in Awaji island, Japan, were conspicuously free of fouling organisms and remain relatively clean. In our laboratory assay, we found that C. sinuosa did not produce any antibacterial compounds. Until now, there are no reports on the role of epibiotic bacteria associated with this alga. In view of this, it would be useful to screen the epibiotic bacterial communities present on algae for their ability to produce QSI compounds or antibacterial activity and to identify them using molecular methods. So, QSI screenings were conducted on several epibiotic bacteria isolated from the surface of C. sinuosa.

For the detection of QSI, McLean et al. (2004) developed a simple protocol based on Chromobacterium violaceum pigmentation inhibition by other bacterial strains. We followed this ‘soft agar overlay protocol’ based on pigmentation inhibition to rapidly screen for the presence of potential QSI by marine bacteria. In our assay, instead of Chr. violaceumwe used Serratia rubidaea JCM 14263 as an indicator organism for the screening of QSI. Some species belonging to Genus Serratia are noted for the production of the red pigment called prodigiosin. In Serratia sp. ATCC 39006, it was reported that the production of prodigiosin was controlled by AHL-mediated QS systems (Thomson et al. 2000). In our recent study (Yamazaki et al. 2006), we found that a strain of Ser. rubidaea JCM 14263 isolated from seawater produces high amount of red pigment, prodigiosin, when growth medium contains 0·5–1 mol l−1 NaCl, and the pigment production was under the control of QS. Hence, we decided this strain as a suitable indicator organism for finding QSI produced by the epibiotic bacteria of C. sinuosa.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Indicator bacteria and algal-epibiotic bacteria

The indicator strain Ser. rubidaea JCM 14263 was isolated from Ariake Sea, a bay located in Kyusyu region, Japan (Yamazaki et al. 2006). As a representative of marine bacteria, two species belonging to the genus Vibrio such as Vibrio fischeri DSM 7151 and Vibrio alginolyticus 138-2 (from H. Tokuda, The University of Tokyo, Tokyo, Japan) were used as a standard test organism in this study. The brown alga, C. sinuosa (Fig. 1), was collected from the intertidal zone of Oiso (34°33′N, 135°0′E) in Awaji Island, Hyogo, Japan. The specimen was thoroughly washed three times with sterile seawater to remove loosely attached bacteria. The surface of seaweeds was swabbed with a sterile cotton tip to obtain epibiotic bacteria. The swab was then used to directly inoculate marine agar plates that contained (per litre) peptone 5 g, yeast extract 1 g, NaCl 30 g, ferric citrate 0·1 g, K2HPO4 0·5 g, NaCl 30 g and agar 15 g (pH 7·5) and incubated at 30°C. Colonies with different morphologies on agar medium were selected. Altogether 96 isolates were obtained from the surface of C. sinuosa. Axenic cultures were obtained by restreaking on agar plates and subsequently stored at 4°C.

image

Figure 1.  Detection of quorum-sensing inhibitors (QSI) by some algal-epibiotic bacteria using Serratia rubidaea JCM 14263 as indicator organism. Soft agar overlay method was used as described in the text. Plates (a) B44, (b) B11, (c) B53 – positive results for QSI produced by epibiotic bacteria; Plate (d) B54 – negative result showing no pigmentation inhibition by unidentified epibiotic bacteria.

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Screening for QSI activity

For the QSI screening, target bacteria were first streaked onto the LB agar plate with appropriate NaCl and incubated overnight. The indicator strain Ser. rubidaea JCM 14263 was grown separately in an LB broth medium. The next day, the target bacterial growth are then covered with an overlay of LB soft agar (full-strength LB broth 0·5% w/v agar), which contains 5 μl inoculum of indicator organism Ser. rubidaea JCM 14263 and then again incubated overnight. Serratia rubidaea itself was used as a control. Vibrio fischeri and V. alginolyticus was used as a positive control. A positive QSI result was indicated by a lack of pigmentation of the indicator strain around the vicinity of the test organisms. Negative results were indicated by no pigmentation inhibition. Photographs were taken after 2 days of incubation using a digital camera (Fig. 1).

Bacterial identification by 16S rRNA sequence analysis

From the selected bacterial colonies, genomic DNA was extracted using phenol–chloroform extraction method. PCR was performed using MicroSeq® 500 16S rDNA Bacterial Identification kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instruction. Positive and negative controls were prepared using Escherichia coli genomic DNA and nuclease-free water, respectively. PCR condition was fixed as initial hold for 10 min at 95°C followed by 30 cycles of melting for 30 s at 95°C, annealing for 30 s at 60°C and extension for 45 s at 72°C. A final extension was fixed for 10 min at 72°C. Using QIAquick PCR Purification kit (Qiagen, UK) according to manufacturer’s instruction, PCR products were purified. Band size and concentration of PCR products were measured by electrophoresis using 2% agarose gel. Purified PCR products were sequenced by using 7 μl of purified PCR products and 13 μl of sequencing modules according to manufacturer’s instruction for each sample. PCR conditions were fixed for 25 cycles (melting 96°C for 10 s, annealing 50°C for 5 s, extension 60°C for 4 min).

After cycle sequencing, excess dye terminators and primers were removed by purification with Clean Seq kit (Agencourt Bioscience Corporation, Beverly, MA, USA), and sequencing was performed using ABI PRISM® 3100 and 3100-Avant Genetic Analyzer (Foster City, CA). The 16S rRNA gene sequences were compared with all other known rRNA gene sequences through a Blast search (http://blast.ddbj.nig.ac.jp/top-j.html). Similar rDNA sequences were downloaded from the database and aligned with our sequences. The ClustalW program (Thompson et al. 1994) was used for preliminary DNA sequence alignment, followed by a manual final alignment. The aligned sequences were subjected to maximum parsimony (MP) analyses in a general heuristic search using paup ver. 4·0b3a (Swofford 1999). Twenty random taxon addition replicates were performed in each heuristic search with the Goloboff fit criterion (k = 2), using the TBR (tree-bisection-reconnection) branch-swapping option. Gaps were considered as missing data in the MP analysis. From the same alignment, two-parameter distances between taxa were estimated, and a phylogenetic tree was constructed with the neighbour-joining (NJ) method, using paup. The robustness of the resulting trees was tested by a bootstrap analysis with 1000 resamplings.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Out of 96 bacteria isolated from the algae, 12% of bacteria were able to produce QSI-like compounds that can be seen from the pigmentation inhibition on Ser. rubidaea JCM 14263 without affecting its growth. The details of the QSI activity measurement was given in Table 1. These 11 bacteria, which were designated as B3, B6, B11, B19, B42, B44, B53, B55, B64, B71 and B74, showed the positive QSI production as can be seen through their pigmentation inhibition, and hence they were selected for the phylogenetic identification using 16S rRNA sequencing method (Fig. 2). The nucleotide sequences of the isolates sequenced in this study have been registered to the DNA Data Bank of Japan (DDBJ)/GenBank with the following accession numbers: B3 (AB361363), B11 (AB361364), B42 (AB361365), B55 (AB361366), B53 (AB361367), B74 (AB361368), B6 (AB361369), B44 (AB361370), B71 (AB361371), B19 (AB361372) and B64 (AB361373).

Table 1.   QSI activity of the bacteria isolated from brown alga Colpomenia sinuosa against pigment-producing bacteria Serratia rubidaea JCM 14263
Bacterial codeQSI activity against Ser. rubidaea
  1. +++, wide (3 cm dia); ++, somewhat narrow (1·5–3 cm dia); +, very narrow (1·5 cm dia); QSI, quorum-sensing inhibitors.

B6, B19, B64+
B3, B42, B71, B74++
B11, B44, B53, B55+++
image

Figure 2.  Molecular phylogenetic tree of the algal-epibiotic bacteria isolated from brown alga, Colpomenia sinuosa, based on 16S rRNA sequences. (a) Neighbour-joining (NJ) tree of the 5′ edge of 16S rRNA sequences of 19 species, including 9 newly reported sequences. (b) NJ tree of the 3′ edge of 16S rRNA sequences of 12 species, including two new sequences. Number at nodes represents bootstrap values in percentage terms based on 1000 replicates.

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For phylogenetic characterization, the 5′ end of 16S rRNA sequences of 19 species was compared with the DDBJ database using the Blast program, including 9 newly reported sequences. The aligned sequences comprised 513 sites and contained 158 parsimony-informative nucleotide positions. In MP analysis, six maximum parsimonious (209 steps, CI = 0·9569, RI = 0·9881) trees were found. Tree topologies were essentially the same for MP and NJ analyses. Molecular phylogenetic analysis demonstrated bacterial isolates showing QSI activities belong to the Bacillaceae (Firmicutes), Pseudomonadaceae (Proteobacteria) and Pseudoalteromonadaceae (Proteobacteria). NJ analysis of 16S rRNA sequences revealed that the strains B3, B11, B42 and B55 belong to Bacillus pumilus, the strain B53 belongs to Bacillus amyloliquefaciens or Bacillus subtilis, the strain of B74 belongs to the Bacillus licheniformis, the strain B6 belongs to Pseudomonas and the strains B44 and B71 belong to Pseudoalteromonas (Fig. 2a). Strains of B3, B11, B42 and B55 showed 99·8–100% similarity (0–1 bp difference) to B. pumilus. Strain B53 showed 100% similarity to B. amyloliquefaciens and 99·8% similarity (1 bp difference) to B. subtilis. Strain of B74 showed 100% sequence similarity to B. licheniformis. The isolate B6 showed 99·6% similarity (2 bp difference) to Pseudomonas stutzeri and Pseudomonas fragi. The strains of B44 and B71 showed 99·6–99·8% similarity (1–2 bp difference) to Pseudoalteromonas haloplanktis and Pseudoalteromonas agarovorans.

The 3′ edge of 16S rRNA sequences of 12 species was compared with the DDBJ database using the BLAST program, including two newly reported sequences. The aligned sequences comprised 745 sites and contained 32 parsimony-informative nucleotide positions. In MP analysis, two maximum parsimonious (90 steps, CI = 0·8222, RI = 0·7714) trees were found. Tree topologies were essentially the same for MP and NJ analyses. Molecular phylogenetic analysis demonstrated bacterial isolates B19 and B64 with antibacterial activity belong to the Vibrio, Vibrionaceae (Proteobacteria) (Fig. 2b). Both isolates showed 100% similarity to Vibrio campbellii, Vibrio harveyi and Vibrio gallicus and 99·9% similarity (1 bp difference) to Vibrio rotiferianus.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

For screening of QSI, earlier studies have used Pseudomonas aureofaciens and Chr. violaceum as an indicator organism (McLean et al. 2004). Pigments of purple-coloured violacein in the case of Chr. violaceum (McLean et al., 1997) and orange-coloured phenazine (Wood and Pierson 1996) in the case of Ps. aureofaciens are effective indicators for QSI produced by terrestrial or nonhalophilic aquatic bacteria. For screening QSI produced by marine bacteria, however, these two strains are not suitable, because they cannot grow or produce pigment in medium with high NaCl concentrations equivalent to sea water. Hence, a strain which can tolerate and produce pigments even in saline conditions could be considered as a suitable indicator for finding QSI from marine/halophilic bacteria. Based on Ser. rubidaea JCM 14263 possessing the abilities such as high salt tolerance together with pigment production, we have attempted to use this strain as an indicator to find QSI from algal-epibiotic bacteria.

For the detection of QSI-like compounds, first we used a set of Vibrio species as test organisms and Ser. rubidaea JCM 14263 itself as control. Positive results of QSI-like compounds (pigmentation inhibition without killing the cells) were produced by V. fischeri and V. alginolyticus. These QSI-like compounds thus have the potential to suppress or inactivate QS-regulated gene expression in target strains which can be seen through the pigmentation inhibition. Negative result for QSI was indicated by noninhibition of pigment production. Serratia rubidaea JCM 14263 strain itself, which was used as a control, did not produce QSI against its own AHL signal mechanism. Among the test organisms used in this study, QS system of V. fischeri has been extensively studied (Lupp and Ruby 2004). Although the compounds responsible for the pigmentation inhibition produced by these Vibrio species have not been identified in this study, their effects are specific to QS-regulated gene expression. From this result and from earlier reports, it is evident that several bacteria belonging to Vibrio species can produce QSI or similar compounds.

In the next set of experiment, QSI screenings were conducted on 96 algal-epibiotic bacteria isolated from C. sinuosa. Some of the bacteria which showed positive and negative QSI activity were shown in Fig. 1. Because it is possible that these bacteria may also produce antibacterial compounds, the pigment inhibition assay should be interpreted with care. The white zone of inhibition around the target strain observed should be opaque and not transparent, i.e., antibacterial agents will inhibit the growth leading to a clear zone of inhibition, whereas QSI activity will permit growth, but inhibit only the formation of pigmentation. Experiments were also conducted to determine whether the methanolic extracts of C. sinuosa have any antibacterial or QSI activity against Ser. rubidaea JCM 14263 by using paper disc assay at a concentration of 30 μl per disc, but it did not showed any activity. From this result, it is clear that this brown algae have no chemical defensive mechanism against fouling or settling bacteria. Algae-epibiotic bacteria have to compete with complex communities of micro-organisms to colonize and persist on algae. It was reported that some bacteria were found to be capable of producing AHL-like molecules to disrupt QS in other bacteria as a means of competition. The green alga, Ulva lactuca relies on the epibiotic bacterium Pseudoalteromonas tunicata to block biofilm formation by the synthesis of pigmented substances that inhibit AHL-dependent transcriptional control (Egan et al. 2002). Bacteria isolated from the surface of the seaweeds have been shown to release compounds that repel other fouling bacteria, suggesting they may protect the seaweed from fouling by other organisms (Boyd et al. 1999; Burgess et al. 1999). From our results on the inhibition activities, it seems that these algal epibiotic bacteria may play a role in the defensive mechanism for their host by producing the QSI or QSI-like compounds to suppress the settlement of competitive bacteria.

The majority of the genus identified in this study, such as Bacillus sp., Pseudoalteromonas sp., Pseudomonas sp., and Vibrio sp., were belonged to common invertebrate-associated bacterial genera (Kanagasabhapathy et al. 2006). Several authors have suggested enzymatic degradation of AHLs as a strategy employed by several bacteria including Bacillus species, in minimizing the detrimental effects caused by QS-regulated products (Dong et al. 2002; Lee et al. 2002). These Gram-positive bacteria secrete an enzyme lactonase that degrades Gram-negative quorum-sensing chemicals by hydrolysing the AHL ring. Likewise, Ps. aeruginosa, a Gram-negative bacterium, has an AHL autoinducer that degrades into tetramic acid, which kills Gram-positive competitors (Sarah 2006). In another study, bacterium Vibrio sp. isolated from the surface of the seaweed Ulva reticulata inhibited the growth of benthic diatoms (Dobretsov and Qian 2002). Genus Pseudoalteromonas is widely retrieved from marine microfouling communities associated with eukaryotic hosts; it is known to produce many bioactive compounds that help to compete for nutrients and for the colonization of surfaces (Holmstrom and Kjelleberg 1999; De Rosa et al. 2000; Egan et al. 2001). From the above results, it seems that the ability to antagonize AHLs seems to be widely distributed in many of the bacterial genus. These bacteria might block the QS systems of their bacterial competitors to obtain a selective advantage over them and may provide the host algae with a tool to control biofouling. Further in-depth studies are required to isolate and identify specific compounds that can elicit the pigment production in these target bacteria.

In this study, we found that several bacteria isolated from brown algae were capable of producing QSI-like compounds that can be seen by their ability to inhibit the pigment production without affecting the cell growth of Ser. rubidaea JCM 14263. An appreciable percentage of bacteria producing QSI-like compounds suggest that these bacteria may prevent the biofilm formation caused by fouling bacteria, which leads to macrofouling on the surface of macroalgae. For primary screening, this simple screening strategy using the pigment inhibition on Ser. rubidaea JCM 14263 strain allows the rapid and sensitive detection of potential QS blockers from large number of bacteria living in marine environments. These QSI compounds could find applications as natural antifoulants in the near future. This study reveals new avenue for future environmental applications.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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