Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

CORALS AS SOURCE OF WITH ANTIMICROBIAL ACTIVITY

Ocky Karna Radjasa1,2*, Jutta Wiese3, Agus Sabdono1,2 and Johannes F Imhoff3

Department of Marine Science, Diponegoro University, Semarang - 50275, Central Java, Indonesia Center for Tropical Coastal and Marine Studies, Diponegoro University, Widya Puraya, Semarang - 50275, Central Java, Indonesia Leibniz-Institute of Marine Sciences, IFM-GEOMAR, Düsternbrooker Weg 20, 24105 Kiel, Germany

Received : February, 22, 2008 ; Accepted : April, 25, 2008

ABSTRACT

In this study we examined marine bacteria associated with different corals (Porites lutea, fascicularis, Acropora sp. and Pavona sp.) collected from vicinity of Panjang island, Jepara, North Java Sea, Indonesia for their antimicrobial activities against the bacteria Echerichia coli, Bacillus subtilis, Staphylococcus lentus and the yeast Candida glabrata. A total of 13 bacterial isolates belonged to the members of Bacillus, Vibrio, Micrococcus, Pseudoalteromonas, Arthrobacter and Pseudovibrio were found to inhibit the growth of at least one test strain. Further examinations among the biologically active strains by using PCR with specific primers of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) resulted in the presence of NRPS gene fragments in the 2 members of Bacillus and Micrococcus and PKS gene fragments in the 2 members of Bacillus and Vibrio. Following cloning and sequencing of the PCR products, the fragments from Bacillus BM1.5 and Micrococcus BJB showed sequence identity with peptide synthetase genes of Bacillus subtilis (61 %) and Actinoplanes teichomyceticus (62.4%). On the other hand, PKS-amplifying strains Bacillus BJ.7 and Vibrio MJ.5 showed closest sequence identity with polyketide synthase genes of Bacillus subtilis (73%) and Anabaena sp 90 (62%), respectively.

Keywords: Coral associated bacteria, Antimicrobial activity, PKS, NRPS

*Correspondence: Phone: +62-24-7460038; Fax: +62-24-7460039; e-mail : ocky [email protected]

INTRODUCTION

Marine invertebrates that are mainly it is important to highlight the possible role accumulating within ecosystems of bacteria in bacteria-invertebrate such as soft corals, sponges, tunicates, and associations in the production of bryozoans have long been recognised as the biologically active substances. sources of structurally unique natural On the other hand, it has been well products. However, it has been suggested known that corals harbor diverse microbial that natural products from marine communities (William et al. 1987; Shashar invertebrates have stricking similarities to et al. 1994; Kim, 1994; Santavy et al. 1995; metabolites of their associated Kushmaro et al. 1996; Rohwer et al. 2001). microorganisms including bacteria (Proksch Every surface immersed in the marine et al. 2002; Imhoff and Stöhr, 2003). Thus, environment, including those organisms 121 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

(Armstrong et al. 2001) such as muco- polypeptide synthetases of the bacterial polysaccharides-covered coral surfaces isolates. providing a nutrient rich habitat for heterotrophic bacteria that leading to the formation of biofilm-forming microbial ATERIALS AND ETHODS communities (Kushmaro et al. 1997). M M Inhibitory interactions among coral- associated bacteria that occur on the coral Sampling and isolation of coral-associated surface then could be of great interest to bacteria search for secondary metabolite-producing bacteria. In addition, examination concernin Four different corals were collected from the secondary metabolite-producing bacteria vicinity of Panjang island, Jepara, North among coral colonizers have been strongly Java Sea, Indonesia by scuba diving and neglected in comparison to soft-bodied identified as Porites lutea, Galaxea marine invertebrates. fascicularis, Acropora sp. and Pavona sp. Polyketides and non-ribosomal Upon collection coral fragments were put peptides are two of the largest groups of into sterile plastic bags (Whirl-Pak, Nasco, multifunctional proteins that create a USA) and immediately brought to the multitude of secondary metabolites Marine Station of the Diponegoro University (Hutchinson, 2003), many of them are used where it was rinsed with sterile seawater and as therapeutic agents et al. 2001; Piel et al. scraped off with a sterile knife. The resultant 2003). Products of the microbial non- tissues were serially diluted in sterile ribosomal peptide synthesis include the seawater, spread on Marine Broth, (Difco, immunosuppressant cyclosporine and other Becton Dickenson GmbH, Heidelberg, antibiotics such as gramicin S, tyrocin A and Germany) and incubated at 20°C for 48 surfactins (Kleinkauf and von Doehren, hours. On the basis of morphological 1996). Among clinically important features, colonies were randomly picked and polyketides are the antibiotibiotic purified by making streak plates (Madigan et daunorubicin, erythromycin, lovastatin and al. 2000). rapamycin (Due et al. 2001). With advanced techniques of Inhibitory interaction tests molecular biology such as polymerase chain reaction (PCR), it is now become possible to Inhibitory interaction tests of coral- carry out a screening on the presence of associated bacteria against test bacteria were polyketides and non ribosomal peptides by performed by using an overlay method. The using specific primers of polyketide following bacteria were used: Escherichia synthases (PKS) (Piel, 2002) and non coli (DSM 498), Staphylococcus lentus ribosomal polypeptide synthetases (NRPS) (DSM 6672), Bacillus subtilis (DSM 347), (Marahiel et al. 1997). and Candida glabrata (DSM 6425) obtained The present study focusses on marine from the German Culture Collection bacteria, which are associated with the (DSMZ, Braunschweig, Germany). corals representing different life forms Overnight cultures of each target microorganism in the logarithmic phase (ca. massive (Porites lutea), sub-massive 9 -1 (Galaxea fascicularis), branching (Acropora 10 cells ml ) were mixed with TSB soft sp.) and folious (Pavona sp.) and produce agar medium (TSB 3g, NaCl 10g and Bacto- secondary metabolites with antimicrobial agar 8g per liter). This mixture (1% v/v, activity. The examinations are coupled with except C. glabrata of 10% v/v) was poured PCR-based analysis for the presence of on to the respective TSB medium surface, polyketide synthases and non-ribosomal which was previously inoculated with coral- 122 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

associated bacteria and incubated for 4 d at PCR amplification and sequencing of 16S 20°C. The plates were then incubated at rRNA gene fragments. 20°C for 48 hours. Antimicrobial activity was defined by the formation of inhibition PCR amplification of partial 16S rRNA gene zones around the bacterial colonies. of active strains, purification of PCR products and subsequent sequencing PCR-based analysis of NRPS and PKS analysis were performed according to the producing bacterial strains method of Thiel and Imhoff (2003). The almost complete 16S rDNA sequences of To obtain genomic DNA of secondary strains were compared for homology to the metabolite producing-strains for PCR NCBI GenBank and EMBL databases using analysis, cell materials were taken from an BLAST (Basic Local Alignment Search agar plate, suspended in sterile water Tool) and FASTA searches (Altschul et al., (Sigma, Germany) and subjected to five 1990, Pearson 1990). Sequences were cycles of freeze (-80°C) and thaw (95°C). aligned using the program ClustalX Version Amplification of peptide synthetase gene 1.83 (Thomson et al. 1997). For fragments was carried out with the NRPS phylogenetic calculations the PhyML degenerated primers A2gamF (5´-AAG software (Guindon and Gascuel, 2003) as GCN GGC GSB GCS TAY STG CC-3´) well as the online version of PhyML and A3gamR (5´-TTG GGB IKB CCG GTS (Guindon et al., 2005) were used. Trees GIN CCS GAG GTG-3´) (Marahiel et al., were calculated by Maximum Likelihood 1997) and PKS degenerated primers (ML) method (Felsenstein, 1981) using the KSDPQQF (5´-MGN GAR GCN NWN GTR model and estimated proportion of SMN ATG GAY CCN CAR CAN MG-3´) invariable sites as well as the Gamma and KSHGTGR (5´-GGR TCN CCN ARN distribution parameter. SWN GTN CCN GTN CCR TG -3´) domain (Piel, 2002). All primers were manufactured Cloning and sequencing of (putative) by MWG-Biotech (Ebersberg, Germany). peptide synthetase and polyketide PCR was performed with an domains ProgeneThermal cycler (Techne, Burkhardtsorf, Germany) as follows: 1 µl The amplified PCR-products were purified template DNA, 1 µl of each of the using the High Pure PCR Product appropriate primers, and 23 µl DNA free Purification Kit (Roche Diagnostics, water (Fluka, Sigma-Aldrich Chemie Mannheim, Germany) following the GmbH, Germany) were added to puReTaq manufacturers protocol. The Big Dye Ready-To-Go PCR beads (Amersham Terminator Cycle Sequencing Ready Biosciences Europe GmbH, Germany). The Reaction Kit (Applied Biosystems) was used NRPS-PCR run comprised 40 cycles with for subsequent sequencing on an ABI 310 denaturing conditions for 1 min at 95°C, analyzer (Perkin Elmer Applied Biosystems, annealing for 1 min at 70°C and extension Foster City, USA). for 2 min at 72°C, respectively. Pseudomonas sp. DSM 50117 was used as DNA sequence accession numbers positive control. The amplification of PKS gene fragments included an initial The 16S rRNA gene sequences have been denaturating step at 94°C for 2 min, entered into the GenBank database under the followed by 45 cycles at 94°C for 1 min, sequence accession number AY000000-, the annealing at 55°C for 1 min and elongation putative peptide synthetase and polyketide at 72°C for 2 min. Bacillus subtilis 168 as synthase sequences obtained under utilized for positive control. AM287210-AM287213. 123 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

synthetase genes of Bacillus subtilis and ESULTS AND ISCUSSION R D Actinoplanes teichomyceticus, respectively (Table 3). Results Inhibitory interaction test Discussion

To estimate antimicrobial activity of coral In a study aimed at the search for associated-bacteria, inhibitory interaction antimicrobial compounds from the sea, we tests between test strains and coral investigated bacteria associated with corals associated isolates were carried out. As from North Java Sea, Indonesia. Our shown in Table 1, a total of 13 bacterial attention was focused on the examination of isolates belonging to the genera coral associated-bacteria by using growth Arthrobacter (1 strain), Bacillus (7 strains), inhibition tests followed by PCR-based Micrococcus (1 strain), Pseudoalteromonas approach for the occurrence of gene (1 strain), Pseudovibrio (1 strain), and fragments of non-ribosomal peptide Vibrio (2 strain), were successfully for their synthetase (NRPS) and polyketide synthase inhibitorial effect against at least 1 test (PKS) which are believed to be responsible strain. All 13 coral derived strains inhibited for the biosynthesis of two main family of the growth of the Gram-positive bacteria B. secondary metabolites, polyketides and subtilis. All isolates but one Pseudovibrio peptides. isolate inhibited the second Gram-positive Microbes including bacteria play test strain, S. lentus. Two Bacillus strains crucial ecological roles in many marine showed an antibacterial activity against the ecosystems such as tropical coral reefs. Gram-negative test strain E. coli, and one Unfortunately, studies on bacterial Bacillus isolate showed an antifungal associations with corals have been much less activity against the yeast C. glabrata. extensive (Knowlton and Rohwer, 2003). Considering the intense interaction among Detection of NRPS and PKS gene coral colonizers, in particular for the fragments by PCR competition for space and nutrient (Slattery et al. 2001), coral associated-bacteria have PCR-based analysis using specific primers been considered as potential sources of of non-ribosomal peptide synthetase (NRPS) biologically active compounds (Moore, and polyketide synthase (PKS) revealed that 1999). the coral-associated bacterial strains Bacillus In our study, we show that the corals sp BM1.5 and Micrococcus sp BJB showed Porites lutea, Galaxea fascicularis, a PCR amplificate for NRPS. In addition, Acropora sp. and Pavona sp. offered two other bacterial strains (Bacillus subtilis promising potency as the source of coral BJ.7 and Vibrio coralliitycus MJ.5) associates with biological activities against exhibited a PCR product of PKS.. E. coli, S. lentus, B. subtilis and C. glabrata. A total of 13 bacterial isolates belonging to Putative peptide synthetase and Gram-positive genera, like Bacillus, polyketide sequences. Micrococcus, and Arthrobacter, and to Gram - negative genera, like Pseudoalteromonas, Two PKS-amplifying bacterial strains (BJ.7 Pseudovibrio and Vibrio were found to and MJ.5) had homologies with polyketide inhibit the growth of at least 1 test strain.. It synthase genes from Bacillus subtilis and is interesting to note that 7 out of 13 active Anabaena sp 90 (Table 2), meanwhile the strains belonged to the genus Vibrio that other 2 NRPS-amplifying strains (BM1.5 were isolated from corals Acropora sp (2), and BJB) showed similarity with peptide G. fascicularis (3) and Pavona sp(2) but not 124 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

from P. lutea. Furthermore, Acropora sp was products many of which are clinically found to dominate the existence of active important drugs. For instance, non- strains including Bacillus (2), Micrococcus ribosomal peptides including cyclosporin, (1), Arthrobacter (1) and Pseudovibrio (1). penicillin and vancomycin are synthesized One isolate obtained from the coral by non-ribosomal peptide sythetases (NRPS) Pavona sp. showed closest similarity to (Kleinkauf and von Doehren, 1990). On the Pseudoalteromonas sp. The genus of contrary, polyketides such as daunorubicin, Pseudoalteromonas is widely distributed in erythromycin, lovastin and rapamycin are the marine environment (Alexeeva et al. derived from sequential condensation of 2003) and considered one of the most short carboxylic acids. abundant group of marine -proteobacteria. In the present study, however, only 2 For the survival in such environments, the strains amplified NRPS and 2 other strains members of genus Pseudoalteromonas have amplified PKS gene fragments Bacillus sp been known to develop metabolic pathways BM1.5 showed closest similarity to the that produce biologically active metabolites. genes for peptide synthetase and protein Extensive studies regarding the capability of building protein of Bacillus subtilis. Toh et Pseudoalteromonas to produce diverse al. (2004) reported the occurrence of emetic secondary metabolites were reported toxin of a foodborne pathogen, Bacillus (Austin, 1989; Jensen and Fenical, 1994; cereus that is putatively a product of non- Holmstrom and Kjelleberg, 1999). Further ribosomal peptide synthesis. The presence of work by (Reid et al. 1993; Deng et al. NRPS gene fragment in coral bacterium 1995) showed that Pseudoalteromonas Bacillus BM1.5 supported the fact that other luteoviolacea, owns a non-ribosomal peptide Bacillus specieroduce peptides using non- synthetase, which produces the siderophore ribosomal peptide synthesis (Stachelhaus alterobactin. Radjasa et al. (2007) reported and Marahiel, 1995). Meanwhile another the antibacterial activity of a bacterium bacterium associated with coral Acropora Pseudoaltermonas luteovioalacea TAB4.2 sp, Bacillus subtilis BJ.7 was found to isolated from coral Acropora sp. collected amplify the gene fragment of PKS and from the neighboring sampling site in the showed sequence similarity of 73% to North Java Sea. A novel antimicrobial previously reported polyketide gene of protein was also reported from a marine Bacillus subtilis (acc. no. U11039). Scotti et bacterium strain X153 which was closely al. (1993) studied the potential ability of B. related to P. piscicida (Longeon et al. 2004). subtilis to synthesize polyketides. Furthermore, Sobolevskaya et al. (2005) Micrococcus sp BJB, had sequence successfully isolated the recently decribed similarity to peptide synthetas of Pseudoalteromonas maricaloris KMM 635T Actinoplanes teichomyceticus.a The collected in the Coral Sea at a depth of 10 m members of Micrococcus are heterotrophic that produces a brominated cyclic bacteria distributed in various environments depsipeptides. such as seawater (Tanaka et al. 2001), Hutchinson (2003) mentioned that marine sediments (Zhong et al. 2002) and microorganisms, especially ones that inhabit prawn-rearing water (Phatarpekar et al. marine environments are noted for the 2002). Members of the genus Micrococcus ability to produce a wide range of chemicals have also the potency as producers of known as secondary metabolites devoted to antimicrobial compounds. A marine the property of self-defense, intercellular bacterium Micrococcus MCCB 104 isolated communication and other aspects of from hatchery water demonstrated microbial life. Du et al. (2001) mentioned extracellular antagonistic properties against that polyketides and non-ribosomal peptides Vibrio alginolyticus, V. parahaemolyticus, V. represent the largest group of natural fluviallis, V. nereis, V. proteolyticus, V. 125 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

mediterranei, V. cholerae and Aeromonas sp of polyketides and peptides among coral- (Jayaprakash et al. 2005). Variacin, a associated bacteria should be of interest bacteriocin produced by Micrococcus because of promising applications in the varians inhibited other Gram-posititve development of pharmacological bacteria, but not Gram-negative ones polyketides and peptides. Furthermore, (Pridmore et al, 1996). considering the fact that research on coral- Two antibacterial active isolates of bacteria association has been fairly limited, vibrios, V. coralliitycus MJ.5 and V. the future study on the search for secondary parahaemolyticus MJ.11 were obtained metabolite producers among coral colonizers from coral Porites lutea. Isolate V. should be given prominence. coralliitycus MJ.5 also amplified the gene fragment of PKS and had sequence similarity (62%) to polyketide synthase of CKNOWLEDGEMENTS Anabaena sp90. Polyketide natural products A are common metabolites of blue-green algae (cyanobacteria) (Burdja et al. 2001). This work was partly supported by the grant Neilan et al. (1999) mentioned that from Ministry of Research and Technology, Cyanobacteria produced a myriad array of Indonesia under the competitive research secondary metabolites, including alkaloids, grant scheme (RUT XI) No. polyketides, and non ribosomal peptides, 011.27.SK.RUT.2004. The work was also some of which are potent toxins. Isolate V. part of a research grant provided by German parahaemolyticus MJ.11 inhibited the Center of Execellent within BIOTECmarin. growth of Bacillus subtilis and Ocky Karna Radjasa was research fellow of Staphylococcus lentus. V. parahaemolyticus the Alexander von Humboldt Foundation has been reported as the member of Vibrio, a (AvH) within the Georg Forster Research dominant genus responsible for the observed Fellowship Programme. mortality in prawn-farmings (Lightner, 1988). As far as known it is uncertain, if V. parahaemolyticus is also a pathogen of REFERENCES corals. Cervino et al. (2004) considered that further studies should include the Alexeeva YV, Kalinovskaya NI, Kuznetsova examination to determine if strains of V. TA, Ivanova EP 2003 Impact of parahaemolyticus and V. alginolyticus are cultivation conditions on haemolytic capable of inducing disease signs in activity of Pseudoalteromonas Montastraea spp. issachenkonii KMM 3549T. Lett. Appl. Microbiol 38:38-42

CONCLUSION Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ 1990 Basic local Our study on coral-associated bacteria alignment search tool. J Mol. Biol provides evidence of antimicrobial 215:403-410 properties among coral colonizers. Although in minor numbers, the coral-associated Altschul SF, Madden TL, Schäffer AA, bacteria might be capable of producing Zhang J, Zhang Z, Miller W, Lipman polyketides and peptides, because of the D J 1997 Gapped BLAST and PSI- detection of NRPS and PKS gene fragments BLAST: a new generation of protein as well as their antimicrobial activities. The database search programs. Nucleic discovery of the potential for the synthesis Acids.Res 25:3389-3402

126 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

Armstrong E, Yan L, Boyd KG, Wright PC, Felsenstein J 1981 Evolutionary trees from Burgess JG 2001. The symbiotic role DNA-sequences - a maximum- of marine microbes on living surfaces. likelihood approach. J. Mol. Evol Hydrobiologia .461: 37-4 17:368-376

Cane DE 1997 Polyketide and non- Fenical W 1993 Chemical studies of marine ribosomal polypeptide biosynthesis. bacteria: developing a new source. Chem.Rev 97: 2463-2706 Chem. Rev 93:1673-1683

Cervino JM, Hayes RL, Polson SW, Polson Guindon S, Lethiec F, Duroux P, Gascuel O SC, Goreau TJ, Martinez SJ, Smith 2005 PHYML Online - a web server GW. 2004. Relationship of Vibrio for fast maximum likelihood-based infection and elevated phylogenetic inference. Nucleic. temperatures to Yellow Blotch/Band Acids. Res 33:W557-W559 disease in Caribbean corals. Appl. Environ.Microbiol. 70: 6855œ6864 Guindon S, Gascuel O 2003 A simple, fast, and accurate algorithm to estimate Christiansen G, Dittmann E, Ordorika lV, large phylogenies by maximum Rippka R, Herdman M, Borner T likelihood. Syst. Biol 52:696-704 2001. Nonribosomal peptide synthetase genes occur in most Jensen P.R, Fenical W 2000 Marine cyanobacterial genera as evidenced by microorganisms and drug discovery: their distribution in axenic strains of current status and future potential. In: PCC. Arch. Microbiol 176:452-458 Fusetani N (ed). Drugs from the sea, Basel: Karger, pp 6-29 Coffroth MA 1990. The function and fate mucous sheets produced by reef Kim K. 1994. Antimicrobial activity in coelenterates. Procc The 6th Int Coral gorgonian corals (Coelenterata, Reef Symp 2:15-20. Australia Octocorallia). Coral. Reefs 13:75-80

Coll JC, Sammarco PW 1986. Soft corals: Kiorboe T, Grossart HP, Ploug H, Kam T chemistry and ecology. Oceanus 2003. Microbial dynamics on 29:33-37 particles: colonization, growth, detachment, and grazing mortality of Deng J G, Hamada Y, Shioiri T 1995. Total attached bacteria. Appl. Environ. synthesis of alterobactin-A, a super Microbiol 69:3036-3047. siderophore from an open-ocean bacterium. J Am Chem Soc 117 (29): Kleinkauf H, von Doehren H 1996. A 824-7825 nonribosomal system of peptide biosynthesis. Eur. J. Biochem Du L, Sanchez C, Shen B 2001. Hybrid 236:335-351 peptide-polyketide natural products: biosynthesis and prospects toward Knowlton N,, Rohwer F 2003. Multispecies engineering novel molecules. microbial mutualisms on coral reefs: Metabol. Engineer 3:78-95 the host as a habitat. Am. Nat 162:51- 62 Faulkner DJ 2000 Marine pharmacology. Antonie. Van. Leeuwenhoek. 77: 135- 145 127 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

Kushmaro A, Loya Y, Fine M, Rossenberg Moore BS 1999. Biosynthesis of marine E 1996 Bacterial infection and coral natural products: microorganisms and bleaching. Nature 380:396 macroalage. Nat. Prod. Rep 16:653- 674 Kushmaro A, Rossenberg E, Fine M, Loya Y, 1997. Bleaching of the coral Munro MHG, Blunt JW, Dumdei EJ, Oculina patagonica by Vibrio AK-1. Hickford SJH, Lill RE, Li S, Mar. Ecol. Prog. Ser 147 159-165 Battershill CN, Duckworth AR 1999. The discovery and development of Lightner, DV. 1988. Vibrio disease of marine compounds with penaeid shrimp, p. 42œ47. In C. J. pharmaceutical potential. J. Sindermann and D. V. Lightner (ed.), Biotechnol 70:15-25 Disease diagnosis and control in North American marine aquaculture. Neilan BA, Dittmann, Rouhiainen L, Bass Elsevier, Amsterdam. The RA, Schaub V, Sivonen K, Borner T Netherlands. 1999. Nonribosomal peptide synthesis and toxigenicity of cyanobacteria. J. Longeon A, Peduzzi J, Barthelemy, Corre S, Bacteriol 181:4089-4097 Nicolas J-L, Guyot M 2004. Purification and partial identification Paul JH, DeFlaun ME, Jefffrey WH 1986 of novel antimicrobial protein from Elevated levels of microbial activity in marine bacterium Pseudoalteromonas the coral surface microlayer. Mar. species strain X153. Mar. Biotechnol Ecol. Prog. Ser. 33:29-40 6:633-641 Pascal H, Vacelet E 1981. Bacterial Long R , Azam F 2001 Antagonistic utilization of mucus on the coral reef interactions among marine pelagic of Aqaba (Red Sea). Procc. 4th. Intl bacteria. Appl. Environ. Microbiol Coral Reef Symp. Manila: pp 669-677 67:4975-4983 Pearson WR 1990. Rapid and Sensitive Ludwig W, Strunk O, Klugbauer S, Sequence Comparison with FASTP Klugbauer N, Weizenegger M, and FASTA. Methods. Enzymol. Neumaier J, Bachleitner M, Schleifer 183:63-98 KH 1998. Bacterial phylogeny based on comparative sequence analysis. Piel J 2002. A polyketide synthase-peptide Electrophoresis 19:554-68 synthetase gene cluster from an uncultured bacterial symbiont of Madigan MT, Martinko JM, Parker J, Brock Paederus beetles. PNAS 29:14002- TD,2000. Biology of microorganisms. 14007 Prentice-Hall, Inc., Upper Saddle River, New Jersey 07458 Piel J, Hui D, Fusetani N, Matsunaga S 2003. Targeting modular polyketide Marahiel MA, Stachelhaus T, Mootz HD synthetase with iteratively acting 1997. Modular peptide synthetases acyltransferases from metagenomes of involved in nonribosomal peptide uncultured bacterial consortia. synthesis. Chem. Rev. 97:2651-2673 Environ. Microbiol. 6: 921-927

Messing J 1983. New M13 vectors for Pietra F, 1997. Secondary metabolites from cloning. Methods. Enzymol 10:20-78 marine microorganisms- bacteria, 128 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

protozoa, algae and fungi- incidence of white band disease. achievements and prospectives. J. Nat. Abstr. Gen. Meet. Am. Soc. Microbiol. Prod. 14: 453-464 p. 332

Proksch P, Edrada RA, Ebel R. 2002. Drugs Scotti C, Piatti M, Cuzzoni A, Perani P, from the seas-current status and Tognoni A, Grandi G, Galizzi A, microbiological implications. Appl Albertini AM 1993. A Bacillus Microbiol Biotechnol. 59: 125-134 subtilis large ORF coding for a polypeptide highly similar to Radjasa, O.K., T. Martens., H-P. Grossart., polyketide synthases. Gene 16:65-71. T. Brinkoff., A. Sabdono., and M. Simon. 2007. Antagonistic activity Scudiero DA, Shoemaker RH, Paull KD, of a marine bacterium Monks A, Tierney S, Nofziger TH, Pseudoalteromonas luteoviolacea Thompson JD, GibsonTJ, Plewniak F, TAB4.2 associated with coral Jeanmougin F, Higgins DG 1997. The Acropora sp. J. Biol. Sci. 7(2):239- ClustalX windows interface: flexible 246 strategies for multiple sequence alignment aided by quality analysis Reid RT, Live DH, Faulkner DJ, Butler A tools. Nucleic. Acids. Res. 25:4876- 1993. A siderophore from a marine 4882 bacterium with an exceptional ferric ion affinity constant. Nature. 366:455- Shashar, N., Cohen, Y., loya, Y., and Sar, N. 458 1994. Nitrogen fixation (acetylene reduction) in stony corals: evidence Rohwer F, Breitbart M, Jara J, Azam F, for coral-bacteria interactions. Mar. Knowlton N 2001. Diversity of Ecol. Prog. Ser. 111: 259-264. bacteria associated with the Caribean coral Montastraea franksi. Coral. Silakowski B, Nordsiek G, Kunze B, Blöker Reefs. 20:85-95 H, Müller R 2001. Novel features in a combined polyketide synthase/non- Rohwer F, Seguritan V, Azam F, Knowlton ribosomal peptide synthetase: the N 2002 Diversity and distribution of myxalamid biosynthetic gene cluster coral-associated bacteria. Mar. Ecol. of the myxobacterium Stigmatella Prog. Ser. 243:1-10 aurantiaca Sga15. Chem. Biol. 8:59- 69 Rublee PA, Lasker HR, Gottfried M, Roman MR 1980. Production and bacterial Slattery M, Rajbhandari I, Wesson K 2001. colonization of mucus from the soft Competition - mediated antibiotic coral Briarium asbestinum. Bull. Mar. induction in the marine bacterium Sci. 30 : 888-893 Streptomyces tenjimariensis. Microb. Ecol. 41:90-96 Sammarco PW, Coll JC 1992. Chemical adaptation in the Octocorallia: Sobolevskaya MP, Smetanina OF, Speitling Evolutionary considerations. Mar. M, Shevchenko LS, Dmitrenok PS, Ecol. Prog. Ser 88: 93-104 Laatsch H, Kuznetsova TA, Ivanova EP, Elyakov GB (2005) Controlling Santavy DL, Peters EC, Kozlowski J, production of brominated cyclic Wilkinson S 1995. Characterization of depsipeptides by Pseudoalteromonas the bacterium suspected in the 129 Journal of Coastal Development ISSN : 1410-5217 Volume 11 Number 3, June 2008 : 121-130

maricaloris KMM 636T. Lett. Appl Toh M, Moffitt MC, Henrichsen L, Raftery Micro.biol. 40:243-248 M, Barrow K, Cox JM, Marquis CP, Neilan BA 2004. Cereulide, the Strunk O, Gross O, Reichel B, May M, emetic toxin of Bacillus cereus, is Hermann S, Stuckmann N, Nonhoff B, putatively a product of nonribosomal Lenke M, Ginhart A, Vilbig A, peptide synthesis. J. Appl. Microbiol. Ludwig T, Bode A, Schleifer K-H, 97:992-1000 Ludwig W 1998 ARB: a software environment for sequence data. Thompson JD, GibsonTJ, Plewniak F, http://www.mikro.biologie.tu- Jeanmougin F, Higgins DG 1997.The muenchen.de/pub/ARB. Department ClustalX windows interface: flexible of Microbiology, Technische strategies for multiple sequence Universität München, Munich, alignment aided by quality analysis Germany. tools. Nucleic. Acids. Res. 25:4876- 4882 Thiel V, Imhoff JF 2003 Phylogentic identification of bacteria with Williams WM, Viner AB, Broughton WJ antimicrobial activities isolated from 1987. Nitrogen fixation (acetylene Mediterranean sponges. Biolmol. Eng. reduction) associated with the living 20:421-423 coral Acropora variabilis. Mar. Biol 94:531-535

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