Third International Seminar on Global Health (3rd ISGH) Technology Transformation in Healthcare for a Better Life ISGH 3 | Vol 3. No. 1 | Oktober 2019 | ISSN : 2715-1948
ISOLATION AND IDENTIFICATION OF CORAL REEF BACTERIA POTENTIAL CANDIDATES FOR PRODUCING BIOACTIVE COMPOUND
Maelita Ramdani Moeis1*, Rohmah Nasada Tuita1, Firdha Rachmawati2 [email protected]
1Institut Teknologi Bandung 2Department of Medical Laboratory Technology, School of Health Sciences Jenderal Achmad Yani Cimahi, Indonesia
ABSTRACT
Background: There is an urgent need to develop new drugs to control serious pathogen. Terrestrial origin of bioactive compound and secondary metabolite has been diminished to be studied. Therefore, more exploration of bioactive compound and secondary metabolite from marine environment becomes important. Acropora digitifera is a coral that is widespread in Indo-Pacific. Many microorganisms live associated with coral reef doing their role. Many active compound have been isolated and characterized from marine microorganisms, which potentially to be used for therapeutic purposes. Therefore, this research was conducted as an initial stage of research to obtain bacteria that have the potential to produce bioactive compounds and secondary metabolites. Objectives: Isolation and identification bacteria from coral reef Acropora digitifera Methods: DNA sample Acropora digitifera was amplified using bacterial marker gene, 16S rRNA. 16S rRNA gene was ligated to cloning vector pGEM-T Easy and transformated to E. coli DH5a. The inserted gene in recombinant plasmid was confirmed by PCR. The gene was sequenced by Macrogen, Korea. The sequence was analysed using BLAST (Basic Local Alignment Search Tool) (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) and phylogenetic tree by MEGA6 with parameter (distance-based) Neighbor Joining (NJ) bootstrap 1000. Results:Twelve isolates were isolated. Seven isolates were Pseudomonas. Three isolates identified as Endozoicomonas euniceicola and one isolate identified as Endozoicomonas elysicola. Endozoicomonas was known has antimicrobial activity. One isolate identified as Brevibacterium permense. Many of Brevibacterium strain was known as bacteriocins producers. Conclusions:Several genus and species bacteria can be isolated from coral reef Acropora digitifera. There are Endozoicomonas euniceicola, Endozoicomonas elysicola, Brevibacterium permense and several genus of Pseudomonas
Keywords: Acropora digitifera, Bio-active compound, Marine bacteria, Metagenome, Secondary metabolite.
INTRODUCTION microbial infections (Rohwer, Seguritan, Azam, & Indonesia has a coral reef ecosystem covering Knowlton, 2002). an area of 50,875 km2 which is composed by Bioactive compound and secondary metabolite various coral species. As many as 18% of coral from terrestrial environment has been diminished. species in the world can be found in Indonesia Therefore, marine environment must to be explored (Roza, 2017). The association between for a novel bioactive compound (Bajpai, 2016). The microorganisms and corals is called coral holobiont. condition between marine environment and Coral consists of symbiosis between coral animals, terrestrial are very different. Therefore, bacteria endosymbiotic algae or zooxanthellae and the might produces different bioactive compound diversity of bacterial communities. Like (Nithyanand & Pandian, 2009). microbiomas in humans, the bacterial community Acropora digitifera coral is widespread in on corals has an important role for corals. The role Indo-Pacific. Amed area is a diving tourism area of microorganisms in corals is to carry out the cycle located in Karang Asem Bali. Amed is divided into of carbon, sulfur, nitrogen and other nutrients for 3 locations, namely Jemeluk, Lipah and Japanese coral hosts. Bacteria also play a role in producing Shipwreck. In this area there are various types of secondary metabolites as protection against corals such as Acropora, Ganiostrea, Pavites and
School of Health Sciences Jenderal Achmad Yani Page 362 Jenderal Sudirman Canal Road – Cimahi 40533 Phone: +62-22-6631622 - 6631624 Isolation and Identification of Coral Reef Bacteria Potential Candidates for Producing Bioactive Compound
Montipora. The author uses DNA samples of DNA will grow white on the media while the other Acropora digitifera that sampled from Jemeluk. is blue. This research was conducted as pre-liminary research to identify bacteria from Acropora Confirmation of transformed DNA digitifera that potentially to be explored more on Plasmid was isolated from transforman cell by bioactive compounds and secondary metabolite. alkalyne-lysis (minipreparation) method (Sambrook, 2003). Isolated plasmid was amplified METHODS by PCR using 16S rRNA universal primer. The 16S rRNA genes amplification amplification of 16S rRNA gene was confirmed by DNA sample of Acropora digitifera was running the amplification product in 1% agarose obtained from the research of Pitasari, 2015 gel. Full-length sequencing of the rRNA gene (Pitasari, 2015). The 16S rRNA genes were (about 1500 bp) for all the coral-associated bacterial amplified by PCR with the universal primer 27F isolates was carried out in Macrogen (Seoul, (5’AGAGTTTGATCCTGGCTC-3’) and 16492R Korea). (5’GGTTACCTTGTTACGACTT-3’) (Oligo Macrogen) and GoTaq Green Master Mix Nucleotide sequence analysis (Promega). PCR was performed with the initial The full-length sequences obtained were DNA denaturation step at 95℃ for 3 min, followed matched with previously published sequences by 30 cycles of DNA denaturation at 95℃ for 30 available in NCBI using BLAST. Multiple sequence sec, primer annealing at 45℃ for 30 sec, and primer analysis was carried out using CLUSTALX ver 2.3. extention at 72℃ for 2 min. The PCR products were MEGA6 with Neighbor Joining method were used analyzed by electrophoresis in 1% agarose gel. to construct the phylogenetic tree. To validate the reproducibility of the branching pattern, 1000 Cloning of 16S rRNA genes bootstrap analysis was performed PCR products were purified by GeneAid Gel/PCR DNA Fragments Extraction Kit RESULT (GeneAidTM) according to manufacturer's The results of the transformation showed that instructions. The purified PCR products was cloned there were 67 bacterial colonies growing on agar in pGEM-T Easy (Promega) vector. Ligation was media. There were 34 transformed colonies that are carried out by centrifugation for 10 sec and white. The white colonies were grown on LB broth incubation at 4℃ for 16 hours. media. Twenty six isolates grew while 8 isolates did not grow. Plasmid isolation was carried out on 26 Transformation of ligated 16S rRNA genes isolates. The 16S rRNA gene amplification results Ligated DNA was transformed to competent showed that only 12 isolates were successfully cell E. coli DHα by heat-shock method. The cold inserted by the full-lenght 16S rRNA gene (1500 competent cell was transferred into a cold microtube bp). containing ligated DNA and incubated at 42℃ for Twelve isolates were identified by 16SrRNA 45-50 sec. Microtube was put back on the ice and gene sequencing (see table 1). Sequence analysis should not be shaken. LB media was added into revealed that 92% belonged to the microtube then incubated at 37℃, 150 rpm, for 1.5 Gammaproteobacteria and 8% to the hours. Transformed DNA was selected by growing Actinobacteria. All the bacteria has the identity the cells on the LB agar containing ampisilin, X- more than 90%. Some of the bacteria has the Gal, and IPTG. Cell colonies with transformed identity more than 97% which are closely related to the previous bacterial spesies.
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Isolation and Identification of Coral Reef Bacteria Potential Candidates for Producing Bioactive Compound
Table 1.BLAST Results of the Sequences
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Isolation and Identification of Coral Reef Bacteria Potential Candidates for Producing Bioactive Compound
Phylogenetic analysis of the bacterial isolates in methane cycle, carbon cycle and nitrogen cycle . were constructed based on the isolate that closely (Madigan, 2014) Pseudomonas and related to the other isolate. The result revealed that Endozoicomonas are genera belonging to the class four isolates (p1, p8, p12 and p13) were closely Gammaproteobacteria. Pseudomonas is able to use related to the genus Endozoicomonas, seven isolates organic compound as carbon and energy source. (p3, p7, p16, p22, p25, p31, p33) were closely This genera has an important role on energy cycle related to genus Pseudomonas and one isolate (p23) and bioremediation agent (Madigan, 2014). was closely related to Brevibacterium. Percentage Pseudomonas flourescence is known to produce an identity <95% indicated isolates of different antibiotic called mupirocin that has the potential to species. Likewise with the bootstrap value <70% fight MRSA (Methicillin-Resistant Staphylococcus indicating that the isolate was not closely related aureus) (El-Sayed et al., 2003). Pseudomonas (Hillis & Bull, 1993). p13 isolate is a close relative synxantha has an important role at nitrogen fixation with Endozoicomonas euniceicola while p1 and p8 (Wechter et al., 2002). Pseudomonas are not. p12 is closely related to Endozoicomonas extremaustralis was found at antartica and this elysicola. p7 and p25 are close relative with bacteria can produce polyhydroxybutyrate (López Pseudomonas flourescence while p22 and p33 are et al., 2009). Polyhydroxybutyrate are accumulated not. Even though p16 identity 99%, the query cover in the granules and exhibit thermoplastic and is low. So that, p16 is not a close relative to elastomeric features, which make them promising Pseudomonas synxantha. p13 and p31 are a close industrial material (Catone et al., 2014). relative with Pseudomonas extremaustralis. p23 is Endozoicomonas associate a lot with marine a close relatives with Brevibacterium permense. biota such as cnidarians, molluscs, porifers, annelids and fish. This genus is well-known as coral symbiont that globally spread from the Great DISCUSSION Barrier Reef in Australia, Papua New Guinea, Gammaproteobacteria is a Gram negative Indonesia to the red sea (Bayer et al., 2013). The bacteria that dominate the habitat of both land and role of Endozoicomonas in its host is not well sea. Gammaproteobacteria is known to be involved known. It is suspected that these bacteria play a role
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in the carbon, sulfur and nitrogen cycles, Characterization of the mupirocin biosynthesis gene synthesizing amino acids, and regulating bacterial cluster from Pseudomonas fluorescens NCIMB colonization at the host (Neave, Apprill, Ferrier- 10586. Chemistry & Biology, 10(5), 419–430. Pagès, & Voolstra, 2016). Endozoicomonas may Hillis, D. M., & Bull, J. J. (1993). An Empirical Test of also produce antimicrobial compounds to deter Bootstrapping as a Method for Assessing Confidence in Phylogenetic Analysis. Systematic Biology, 42(2), invading pathogenic microbes, which has been seen 182–192. https://doi.org/10.1093/sysbio/42.2.182 for other coral-associated bacteria (Neave, Michell, López, N. I., Pettinari, M. J., Stackebrandt, E., Tribelli, Apprill, & Voolstra, 2017). P. M., Põtter, M., Steinbüchel, A., & Méndez, B. S. Actinobacteria is a group of bacteria with the (2009). Pseudomonas extremaustralis sp. nov., a Poly characteristics of rod-shaped, Gram-positive, (3-hydroxybutyrate) producer isolated from an aerobic, has a high G + C content and is found in antarctic environment. Current Microbiology, 59(5), many soils, freshwater and seawater. Actinobacteria 514–519. can use organic compounds and play a role in the Madigan, et al. (2014). Brock Biology of carbon cycle (Madigan, 2014). Nithyanand & Microorganisms 15th edition (fifteenth). NY: Pandian, 2009 reported that Actinobacteria such as Pearson Education. Neave, M. J., Apprill, A., Ferrier-Pagès, C., & Voolstra, Brevibacterium sp. exhibited antibacterial activity C. R. (2016). Diversity and function of prevalent against various pathogens (Nithyanand & Pandian, symbiotic marine bacteria in the genus 2009). According to the literature, this coral- Endozoicomonas. Applied Microbiology and associated bacteria has a great potential to be Biotechnology, 100(19), 8315–8324. explored more for bioactive compound in the next https://doi.org/10.1007/s00253-016-7777-0 research. Neave, M. J., Michell, C. T., Apprill, A., & Voolstra, C. R. (2017). Endozoicomonas genomes reveal CONCLUSION functional adaptation and plasticity in bacterial Several genus and species bacteria can be strains symbiotically associated with diverse marine isolated from coral reef Acropora digitifera. There hosts. Scientific Reports, 7(October 2016), 1–12. https://doi.org/10.1038/srep40579 are Endozoicomonas euniceicola, Endozoicomonas Nithyanand, P., & Pandian, S. K. (2009). Phylogenetic elysicola, Brevibacterium permense and several characterization of culturable bacterial diversity genus of Pseudomonas. Some of the genus were associated with the mucus and tissue of the coral known having antimicrobal activity. Coral- Acropora digitifera from the Gulf of Mannar. FEMS associated bacteria needs to be explored more for Microbiology Ecology, 69(3), 384–394. bioactive compounds. https://doi.org/10.1111/j.1574-6941.2009.00723.x Pitasari, A. (2015). Keragaman Genetik Acropora REFERENCES digitifera di Kawasan Amed, Bali. Institut Teknologi Bajpai, V. K. (2016). Antimicrobial bioactive Bandung. compounds from marine algae : A mini review. Rohwer, F., Seguritan, V., Azam, F., & Knowlton, N. Indian Journal of Geo-Marine Scieences, 45(9), (2002). Diversity and distribution of coral-associated 1076–1085. bacteria. Marine Ecology Progress Series, 243, 1–10. Bayer, T., Arif, C., Ferrier-Pagès, C., Zoccola, D., Roza, E. (2017). Maritim Indonesia, Kemewahan Yang Aranda, M., & Voolstra, C. R. (2013). Bacteria of the Luar Biasa. Retrieved from genus Endozoicomonas dominate the microbiome of http://www2.kkp.go.id/awid1/artikel/2233-maritim- the Mediterranean gorgonian coral Eunicella indonesia-kemewahan-yang-luar-biasa cavolini. Marine Ecology Progress Series, 479, 75– Sambrook, J. (2003). Molecular cloning : a laboratory 84. manual. Retrieved from Catone, M. V., Ruiz, J. A., Castellanos, M., Segura, D., https://search.library.wisc.edu/catalog/99989792460 Espin, G., & López, N. I. (2014). High 2121 polyhydroxybutyrate production in Pseudomonas Wechter, W. P., Begum, D., Presting, G., Kim, J. J., extremaustralis is associated with differential Wing, R. A., & Kluepfel, D. A. (2002). Physical expression of horizontally acquired and core genome mapping, BAC-end sequence analysis, and marker polyhydroxyalkanoate synthase genes. PLoS ONE, tagging of the soilborne nematicidal bacterium, 9(6). https://doi.org/10.1371/journal.pone.0098873 Pseudomonas synxantha BG33R. Omics: A Journal El-Sayed, A. K., Hothersall, J., Cooper, S. M., Stephens, of Integrative Biology, 6(1), 11–21. E., Simpson, T. J., & Thomas, C. M. (2003).
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