Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is.

ANTIMICROBIAL ACTIVITY OF BIOACTIVE COMPOUNDS

ISOLATED FROM MARINE BACTERIA ASSOCIATED WITH

SPONGE

Jaspis

sp. AND THEIR GENETICS ANALYSIS

EFFENDI

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

DECLARATION

I hereby declare that this thesis entitled “Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. an d Their Genetics Analysis” is the result of my own work through the guidance from my academic supervisors and has not been submitted in any form for another de gree at any other uni versity. Sources of information de rived or quoted from published and unpublished works of ot her authors is mentioned in the text and listed in the list of References at the end o f this thesis.

Bogor, August 2012

Effendi G351100021

ABSTRACT

EFFENDI. Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is. Under direction of ARIS TRI WAHYUDI and MUNTI YUHANA.

Antimicrobial compounds of three marine bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 which associated with sponge Jaspis sp. were extracted using ethyl acetate solvent. Each of bacterial crude extract showed different activity against non-pathogenic microbes i.e. Bacillus subtilis, Escherichia coli, and pathogenic microbes i.e. Staphylococcus aureus, enteropathogenic E. coli K1-1 (EPEC K1-1), Pseudomonas aeruginosa, Candida albicans and C. tropicalis. Bacterial crude extract of isolate SAB E-41 demonstrated the best antimicrobial activity against non-pathoge nic and pathogenic microbes. Active fractions of each bacterial crude extract were detected using bioautography method. Fractionation and purification of antimicrobial compound for bacterial crude extract from isolate SAB E-41 was carried out using silica gel-column chromatography and preparative thin layer chromatography (PTLC) technique. Analysis of 16S rDNA for those three isolates showed that they were included in the genus of Bacillus. Sequence analysis of cloned DNA fragments encoding ketosynthase (KS) do main showed that they had homology with PKS type I of B. amyloliquefaciens LL3 for SAB E-41 and putative polyketide synthase pksL of B. amyloliquefaciens subsp. plantarum CAU-B946 for SAB E-57. The adenylation (A) domain of SAB E-31 showed its homology with bacitracin synthetase I of B. pumilus ATCC 7061, whereas for SAB E-41 and SAB E-57 showed their homology to sur factin synthetase B of B. amyloliquefaciens subsp. plantarum CAU-B946 and surfactin synthetase A of B. amyloliquefaciens subsp. plantarum CAU-B946, respectively.

Keywords: Antimicrobial compound, fractionation, cloning, 16S rDNA, KS and A domain, Jaspis sp.

SUMMARY

EFFENDI. Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is. Under direction of ARIS TRI WAHYUDI and MUNTI YUHANA.

The increase of global resistance of the pathogenic microbes against various antibiotics becomes a serious concern in public health. Many efforts are conducted in order to solve this problem, such as finding the new bioactive compounds from marine bacteria which associated with marine sponges. Three marine bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that associated with sponge Jaspis sp. showed their capability in producing antimicrobial compounds. In this study, extraction of antimicrobial compounds from these bacteria was conducted using ethyl acetate solvent. Each of bacterial crude extract displayed significant activity against B. subtilis, E. coli as non-pathogenic microbes and S. aureus, P. aeruginosa, enterop athogenic E. coli K1-1, C. albicans and C. tropicalis as pathogenic microbes. Bacterial crude extract of isolate SAB E-41 demonstrated better activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57.

Analysis of constituent component for each bacterial crude extract was conducted using thin layer chromatography (TLC). Each of bacterial crude extract was spotted onto silica gel plate and eluted with n-butanol-ethyl acetate solvent mixture (3:7). Six spots/fractions were successfully detected by viewing under UV light at 254 nm and 366 nm wave- length. Active spots/fractions from each bacterial crude extract were detected using bioautography method. Four spots from each bacterial crude extract showed antimicrobial activity against P. aeruginosa and t wo spots showed antimicrobial activity against S. aureus.

Fractionation of bacterial crude extract from isolate SAB E-41 was carried out using silica gel-column chromatography. Two hundred and five fractions were successfully collected from fraction collector and combined into thirty compos ite fractions based on the same chromatogram by using TLC ana lysis. The antimicrobial activity of thirty compos ite fractions was tested to S. aureus, P. aeruginosa, enteropathogenic E. coli K1-1, C. albicans and C. tropicalis. F ifteen of the m named by 1, 2, 3, 4, 5, 6, 7, 8, 11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 have different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and C. albicans. Fraction BA-2 that was eluted by chloroform- methanol (90% -10%) solvent system showed antifungal activity against C. albicans while fraction BA-13 that was eluted with chloroform-methanol (50% -50%) solvent system showed the highest inhibition against S. aureus followed by fraction BA-17. The diameter of inhibition zone that formed by these two active fractions were about 12 mm and 14 mm. Fraction BA-2 and BA-4 that was eluted with chloroform- methanol (90%-10%) showed the best activity against enteropathogenic E. coli K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm.

Purification of fifteen compos ite fractions was conducted using preparative thin layer chromatography (PTLC) technique. Four active fractions with the Rf values of 0.87; 0.50; 0.41 and 0.12 were successfully collected from fraction

BA-13 and displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with the Rf values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18. Bot h of these active compounds were successfully collected and showed antimicrobial activity against S.

Morphological and molecular identification of those three isolates were carried out in order to identify these bacteria. Sequences analysis of 16S rDNA showed that three isolates were included in the genus of Bacillus. Isolate SAB E-31 had 98% of homology level with B. pumilus strain KD3 while isolate SAB E-41 had 98 % of homology level with B. amyloliquefaciens strain zy2 and isolate SAB E-57 had 97% of homology level with B. subtilis strain YRL02.

aureus.

PCR amplification of ketosynthase (KS) domain of PKS and adenylation (A) domain of NRPS were successfully amplified and sub-cloned into T-Vector pMD20. All isolates coded as SAB E-31, SAB E-41 and SAB E-57 possessed A domain and only two isolates coded as SAB E-41 and SAB E-57 possessed KS domain. DNA fragment encoding KS domain was in a size of 700 bp while for A domain was in a size of 1000

Sequences analysis of DNA fragment encoding KS domain using BlastX program indicated that isolate SAB E-41 showed a similarity level of 97% with type I PKS from Bacillus amyloliquefaciens LL3 and isolate SAB E-57 showed a similarity level of 98% with putative polyketide synthase pksL from B. amyloliquefaciens subsp. plantarum CAU-B946 whereas for A domain indicated that isolate SAB E-31 showed a similarity level of 81% with bacitracin synthetase 1 from B. pumilus ATCC 7061. Isolate SAB E-41 s howed a similarity level of 80% with sur factin synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 and isolate SAB E-57 showed a similarity level of 81% with surfactin synthetase A from the same strain of Bacillus, CAU-B946.

bp.

Keywords: Antimicrobial compound, fractionation, cloning, 16S rDNA, KS and A domain, Jaspis sp.

Copyright © 2012, by Bogor Agricultural University Copyright are protected by law

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ANTIMICROBIAL ACTIVITY OF BIOACTIVE COMPOUNDS

ISOLATED FROM MARINE BACTERIA ASSOCIATED WITH

SPONGE

Jaspis

sp. AND THEIR GENETICS ANALYSIS

EFFENDI

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Microbiology Study Program

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

Title : Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analysis

Name : Effendi Student ID : G351100021

Approved Advisory Committee

Dr. Aris Tri Wahyudi, M.Si

Chairman Committee Member

Dr. Munti Yuhana, M.Si

Agreed

Coordinator of Microbiology Mayor Dean of Graduate Schoo l

Dr. Ir. Gayuh Rahayu Dr. Ir. Dahrul Syah, M.Sc. Agr.

ACKNOWLEDGEMENTS

First and foremost, I would like to thanks God for the health, grace and gifts so this thesis can be completed on time. The topic for this thesis was “Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analysis.

I would like to say gratefully thanks to Dr. Aris Tri Wahyudi, M.S i as the main supervisor who gave me the opportunity to work on this truly exciting research project and Dr Munti Yuhana, M.Si as the committee member. I wish to express my sincere thanks and gratitude for their guidance, great support, encouragement, patience and scient ific knowledge that were given to me. Thank you very much also to Dr. Ir Ence Darmo Jaya Supena, M.S as the head of Department of Biology and Dr. Ir Widanarni, M.S i as the examination committee that would like to give their suggestion for the improvement of this thesis.

Gratefully thanks also to Prof. Masafumi Yohda and Prof. Masafumi Odaka which gave the permission for me and all of the facility during the research in the laboratory of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Japan. Gratefully thanks also to Prof. Wuled Lenggoro which supported me during I followed the Short Stay/Shor t Visit (SSSV) program in Japan. Thank you very much to Neng Risma Liasari and all of the stude nts in Yohda Lab. and Lenggoro Lab. that would like to share about their scientific knowledge and helped me during the research in Japa n. Thank you also to head of laboratory, all of the staff and stude nts in the labo rator y of Microb iology and Biopharmaca Research Center, IPB, Bogor for their research’s permission and helpful effort. Finally, I would like to say gratefully thanks to my parents and brothers that already supported me during I study at Bogor Agricultural University. “This work is dedicated to my lovely parents and my best brothers”.

Bogor, August 2012

BIOGRAPHY

The author was born in Medan on August 21st

In 2010, the author was accepted as a Graduate student at Bogor Agricultural University, especially in Microb iology Study Program. During the study, the author worked at PT. Mitra Pelajar as an instructor of Biology for 4 months. In 2011, the author was accepted as a participant in the Short Stay/Short Visit (SSSV) program for 3 months which was held by Tokyo University of Agriculture and Technology (TUAT), Naka-cho, Koganei, Tokyo, Japan. On June 6

, 1987 as the third son from Mr. Tan Po Tiam (R.I.P) and Mrs. Ong Siu Ing. In 2005, the author completed his senior high school at Yayasan Perguruan Tinggi Sutomo 2, Medan and in the same year, the author was accepted as an undergraduate student at University of Sumatera Utara, especially in Biology Study Program. During the study, the author was active at Microb iology Study Club (MSC) organization that held by Department of Biology. Beside of that, the author also worked as a Lab. Assistant in the laboratory of Microbiology and the laboratory of Plant Structure and Develop ment. In 2009, the aut hor finished the unde rgraduate schoo l and got the Bachelor of Science degree. After finished the study, the author worked as a private teacher for one year.

th

– 8th, 2012, part of this thesis with the title “Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp.” has been presented in the International Seminar on Advances in Molecular Genetics and Biotechnology for Public Education hosted by Atma Jaya Catholic University of Indo nesia.

TABLE OF CONTENT

Page

TABLE OF CONTEN T... i

LIST OF TABLES ...ii

LIST OF FIGURES ...iii

LIST OF APPENDIXES ...iv

INTRODUCTION Background ... 1

Aims and Scope of the Study ... 2

LITERATURES Marine Natural Products ... 3

Marine Spo nge-Associated Bacteria ... 4

Bioactive Compounds ... 6

Polyketide Synthase and Nonribosomal Peptide Synthetase ... 8

MATERIALS AND METHODS Duration and P lace of Study ... 11

Materials... 11

Extraction of Antimicrobial Compounds ... 11

Antimicrobial Activity Test ... 12

Detection of Antimicrobial Compounds ... 13

Fractionation of Bacterial Crude Extract ... 13

Purification of Antimicrobial Compounds... 14

Morphological and Molecular Identification ... 14

DNA Extraction ... 14

PCR Amplification of KS and A Domain... 15

Cloning of DNA Fragments Encoding KS and A Domain ... 16

Sequencing a nd Bioinformatics Analysis of KS and A Domain ... 16

RESULTS Antimicrobial Activity of Bacterial Crude Extracts ... 17

Thin Layer Chromatography and Bioautography ... 18

Fractionation of Bacterial Crude Extract from Isolate SAB E-41 ... 20

Purification of Antimicrobial Compounds using Preparative TLC ... 22

Morphological and Molecular Identification Based on 16S rDNA Analysis... 24

Amplification of DNA Fragments Encoding KS and A Domain ... 26

Cloning a nd Bioinformatics Analysis ... 26

DISCUSSION ... 33

CONCLUSION AND SUGGESTION ... 45

REFERENCES... 47

LIST OF TABLES

Page 1 Bacterial isolates identification from marine sponges ... 5 2 Bioactive compounds produced by marine sponges ... 7 3 Diameter average of inhibition zone (mm) from three bacterial

crude extracts (100 mg/ml) produced by sponge-associated bacteria... 17 4 Variation of Rf values from three bacterial crude extracts eluted

with different solvent systems ... 18 5 Active spots/fractions of three bacterial crude extracts

detected using bioautography method ... 20 6 Antimicrobial activity of thirty composite fractions

collected from silica gel-column chromatography... 21 7 Rf values of active compounds from fifteen composite fractions ... 23 8 Similarity of 16S rDNA sequences from isolates SAB E-31

SAB E-41 and SAB E-57 compared with GenBank Database ... 25 9 Bioinformatics sequences analysis of DNA fragments

encoding KS domain using BlastX Program ... 27 10 Bioinformatics sequences analysis of DNA fragments

LIST OF FIGURES

Page 1 Organization of the multimodular PKS gene cluster in

metagenomic libraries from D. dissolute ... 9 2 Two A domains ins ide the biosynthetic gene cluster of

Onnamide B ... 9 3 Flowchart of procedural steps used in this study ... 12 4 Antimicrobial activity of three bacterial crude extracts isolates

SAB E-31, SAB E-41 and SAB E-57 using agar diffusion method ... 18 5 Profile of each bacterial crude extract on silica gel plate merck

60 F254

6 Antimicrobial activity of active spots/fractions using

eluted with n-butanol and ethyl acetate mixture (3:7)... 19

bioautography method ... 19 7 Antimicrobial activity of thirty compo site fractions ... 22 8 Profile of active compounds from fraction BA-13, BA-17 and

BA-18 o n silica gel plate merck 60 F254

9 Antimicrobial activity of fifteen composite fractions purified

... 24

using PTLC Technique ... 24 10 Gram staining o f three bacterial isolates coded as: A). S AB E-31;

B). SAB E-41 and C). SAB E-57... 24 11 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57

with the reference strains ba sed o n 16S rDNA sequences... 25 12 Agarose gel electrophoresis of DNA fragments encoding

KS domain and A Domain ... 26 13 Restriction of recombinant plasmid digested with BamHI + XbaI

A). pMD20-KS Domain and B). pMD20-A Domain ... 27 14 Alignment of amino acid sequences of KS domain from isolates

SAB E-41 and SAB E-57 w ith the reference strains in GenBank

Database using ClustalW program... 29 15 Phylogenetic tree of isolates SAB E-41 and SAB E-57 with

the reference strains based on amino acid sequences of KS domain ... 29 16 Alignment of amino acid sequences of A domain from isolates

SAB E-31, SAB E-41 and SAB E-57 w ith the reference strains

in GenBank Database using ClustalW program ... 31 17 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57

with the reference strains based on amino acid sequences of

LIST OF APPENDIXES

Page 1 The 16S rDNA sequences of three marine bacterial isolates... 55 2 DNA sequences of KS domain of PKS gene from two bacterial

isolates... 57 3 DNA sequences of A domain of NRPS gene from three bacterial

isolates... 58 4 Alignment of 16S rDNA sequences from isolate SAB E-31

using BlastN program ... 60 5 Alignment of 16S rDNA sequences from isolate SAB E-41

using BlastN program ... 62 6 Alignment of 16S rDNA sequences from isolate SAB E-57

using BlastN program ... 64 7 Plasmid map of T-Vector pMD20 (TaKaRa Bio Inc.) ... 66 8 Alignment of DNA sequences encoding KS domain of PKS gene

from isolate SAB E-41 using BlastX program ... 67 9 Alignment of DNA sequences encoding KS domain of PKS gene

from isolate SAB E-57 using BlastX program ... 68 10 Alignment of DNA sequences encod ing A do main o f NRPS gene

from isolate SAB E-31 using BlastX program ... 69 11 Alignment of DNA sequences encoding A domain of NRPS gene

from isolate SAB E-41 using BlastX program ... 70 12 Alignment of DNA sequences encoding A domain of NRPS gene

INTRODUCTION

Background

The improper and uncontrolled uses of antibiotics against pathogenic bacteria induced and accelerated the occurrence of multi drugs resistant (MDR) strain. The number of infection cases by MDR strains in Indonesia remains high. Some of infection cases were tuberculosis, HIV, malaria, diarrhea and infection on upper respiratory system (Ditjen PP & PL Depkes RI 2011). In 2009, Indo nesia was ranked eighth of 27 countries with the highest of multi drugs resistant cases in the world (WHO 2010)

Nowadays, the exploration of bioactive compounds has been carried out in many kinds of resources such as medicinal plants, animals, aquatic organisms as well as microorganisms in unique ecosystem in order to find the new bioactive compounds which can treat the MDR strains. Indonesia was one of the hotspot countries possessing many natural resources and almost 70% of the area was covered by coastal area. Considering that, the exploration of new bioactive compounds in an aquatic area is very promising for the new inve ntion of

. The increase of MDR cases has encouraged many scientists to find the new bioactive compounds in order to solve the MDR problem.

chemot herape utic agents

Marine sponges are one of the evolutionary multicellular organisms that have been reported very potential in producing many kinds of bioactive compounds. Some of the bioactive compounds showed antibacterial, antifungal, antiviral, anticancer, antifouling and cytotoxic properties (Taylor et al. 2007). The limitation of sponge biomass is the main factor for isolating the large scale of bioactive compounds. Therefore, alternative and ecologically sound sources of bioactive compounds are needed. Marine microorganisms have contributed to the majority of bioactive compounds. They can produce the same metabolite compo unds as their host (Proksch et al. 2002).

which can be developed and applied in pharmaceutical industry in the future.

The surfaces and internal spaces of marine sponges are unique microhabitat and more nutrient rich than seawater or most sediments, thus they would likely be a unique niche for the isolation of diverse microor ganisms (Friedrich et al. 1999).

Isolation and screening the potent bacterial isolates from marine sponge, identifying the antibiotic-encoding genes in active microorganisms, cloning in amenable host and characterize the bioactive compounds were the main strategy for producing large amounts of new metabolites (Webster & Hill 2001).

Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 have been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West Papua Province. Each of these isolates indicated different activity against Staphylococcus aureus, Escherichia coli, enteropathogenic E. coli K1-1, Pseudomonas aeruginosa, Candida albicans and C. tropicalis by using multilayer technique (Abubakar 2009). Extraction, fractionation and purification of antimicrobial compounds for these isolates were important for this study in order to characterize their antimicrobial compounds. Analysis of 16S rDNA and detection of KS domain of PKS and A domain of NRPS genes were important for identifying these bacteria as well as ensuring their capability in synthesizing the bioactive compounds.

Aims and Scope of the Study

The aims of this study were to determine the antimicrobial activity of bioactive compounds, analyze the 16S rDNA and detect the occurrence of KS and A domain of PKS and NRPS genes of those three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that symbiosis with sponge Jaspis sp.

LITERATURES

Marine Natural Products

The ocean covers more than 70% of earth surface and is considered as a great reservoir of natural resources. However, the extent of marine biodiversity, especially of microor ganisms, is barely known. Marine microbial communities are composed of ubiquitous members that can be found not only in the sur face waters of the sea, but also in the lower and abyssal depths from coastal to the offshore regions (Larsen et al. 2005). Several studies have repor ted the discovery of new bioactive compounds from marine organisms, focusing mainly on chemistry of secondary metabolites, which include now more than 15,000 structurally diverse bioactive compounds isolated during the last 30 years (Salomon et al. 2004).

Given the diversity of marine or ganisms and habitats, marine natural products encompass a wide variety of chemical classes such as terpenoid, polyketides, acetogenins, peptides and alkaloids of varying structures representing biosynthetic schemes of stunning variety (Wright 1998). Marine sponges are one of the benthic organisms that play a potential role as natural compounds producer. They also became a host for a wide range of microbes. The role of these diverse microbes in sponge biology varies fromthe digestion of microbes as a food source to mutualisticsymbiosis with the spo nge. On the ot her hand, sponge is believed to provide shelter from predators, a substrate for colonization, access to sunlight for photosyntheticmicrobes and a supply of nutrients (Taylor et al. 2007).

The availability of sponge biomass is the main factor for isolating marine natural prod ucts. Therefore, marine microor ganisms which associated with marine sponges became one of the alternative ways to solve that problem. They have contributed to the majority of marine natural prod ucts and produced the same metabolite compounds as their hos t. Friedrich et al. (2001) reported that many sponges contain enormous amounts of bacteria within their tissues, sometimes occupying 40 to 60% of the total biomass (equivalent to 108 to 1010 bacteria per gram). Considering to the rich diversity of microor ganisms in their tissues and the growth of microor ganisms were more rapidly, therefore isolation and cultivation of

associated microorganism producer of bioactive compounds could help to solve the recognized problem of development of potential sponge-derived drugs.

Convincing evidence for the involvement of microor ganisms in natural product synthesis has been complied for the tropical sponges Dysidea herbacea and Theonella swinhoei, in which the producing microbe is a cynobacterium in the former and a bacterium in the latter (Proksch et al. 2002). Thus an alternative strategy targeting the microor ganisms associated with sponges for the screening of bioactive natural products may prove to be an effective approach to circumvent the associated difficulties of dealing with the organism itself.

Marine Sponge -Associated Bacteria

Sponges are filter feeders animal which live in areas with strong c urrents or wave action. Most carnivorous animals avoid sponges because of the splinter-like spicules and toxic chemicals produced/sequestered by the sponge. Sponges are organized around a system of pores, ostia, canals and chambers that are used to canalize the large flow of water that is pumped through spo nges. The water enters the sponge through the inhalant canals and exits by the oscules. A sponge is constituted of three layers. The first layer comprises pinacocytes and is called the pinacoderm. Under the pinacoderm is the mesohyl region that contains canals and choanocyte chambers. This is where the sponge metabolisms, reproduction and nutrient transfer occur. The third layer is the choa nod erm and contains choanocytes. They are flagellated cells possessing a collar of cytoplasmic tentacles. It is through the movement of these tentacles that the flow of water is created bringing in nutrients (Wilkinson 1992).

There are mainly three classes of sponges, namely the Calcarea (5 orders and 24 families), Demospongiae (15 orders and 92 families) and Hexactinellida (6 orders and 20 families). So far about 15.000 species of sponges have been described, but their true diversity may be higher (Fieseler et al. 2004). Most of the species are placed under the class Demospongiae. Since sponges are simple and sessile organisms; during e volut ion they have de velope d pot ent chemical defensive mechanism to protect themselves from competitors and predators as well as infectious microorganisms (Belarbi et al. 2003).

Interaction between marine sponges and living aquatic microorganisms are so variously and had many important roles. Many microorganisms were found that growth commensally in the surface and also found inside of the other microorganisms such as in the food digestive system. Porus from sponges contain diverse bacteria. Reinheimer (1991) found that there were many kinds of bacteria such as Pseudomonas, Bacillus, Micrococcus, Aeromonas, Vibrio, Achromobacter, Flavobacterium and Corynebacterium in Microcionia prolifera sponge.

There is a symbiotic connection between sponges and a number of bacteria and algae. Sponges give protection for the symbionts and the symbionts give nutrition for sponges. Algae that symbiotic with sponges give nutrient from their photosynthesis product (Taylor et al. 2007). Suryati et al. (2000) reported that the formation of bioactive compounds from sponges was depend on the precursor of enzyme, nutrient and product of symbiotic with another biota that contain bioactive compounds such as bacteria, mold and another kinds of dinoflagellata that can spur on producing bioactive. Suryati et al. (2000) found a number of sponge types living in Spermonde seawater, South Sulawesi, the diversity of mold and symbiotic bacterial with sponges are so variously and usually dominated by Aeromonas, Flavobacterium, Vibrio, Pseudomonas, Acinetobacter and Bacillus (Table 1).

Table 1 Bacterial isolates identification from marine sponges (Suryati et al. 2000)

No. Marine Sponges Bacte rial species

1. Acanthela clethera Flavobacterium sp., Aeromonas sp. 2. Aplisina sp. Aeromonas sp.

3. Callyspongia sp. Pseudomonas sp. 4. Clathria bacilana Aeromonas sp. 5. Clathria reinwardhi Aeromonas sp.

6. Jaspis sp. Flavobacterum sp.

7. Phakelia aruensis Bacillus sp., Aeromonas sp.

8. Phyllospongia sp. Vibrio sp., Pseudomonas sp., Aeromonas sp. 9. Reniochalina sp. Acinetobacter sp.

10. Thionella cilindrica Aeromonas sp.

11. Stylotella aurantiorum Aeromonas sp., Vibrio sp.

12. Xestospongia sp. Enterobacteriaceae sp., Aeromonas sp.

Experimental evidence suggests that there are qualitative and quantitative variations in secondary metabolites produced by some organisms. There is a tende ncy to explain these variations ecologically with environmental factors

influencing the biochemical profile of the organisms. There are many examples of marine invertebrates with chemical defenses. The synthesis and storage of these substances favors the survival of such organisms in a complex environment and gives such species a selective advantage due to the genetic transmission of the capacity for chemical defense synthesis (Chr istop hersen 1991).

Menezes et al. (2010 ) reported that microbial diversity associated with algae, ascidians and sponges from the north coast of Sao Paulo State, Brazil had been dominated by phylum Firmicutes, Bacillus spp., together with Ruegeria spp. Bacillus was the most abundant genus recovered, with 33 isolates, followed by Ruegeria with 31 isolates and Micrococcus with 23 isolates. All of them revealed broad distribution among the marine macroor ganisms sampled. 16S rDNA sequencing-based analysis showed that marine-derived bacteria were related to 41 genera distributed among the phyla Proteobacteria (35.4%), Actinobacteria (30.4%), Firmicutes (28.7% ) and Bacteroidetes (1.1%).

Bioactive Compounds

Marine sponges are pre-eminent producers of bioactive secondary metabolites and their repertoire includes peptides, terpenes and sterols. Many of these compounds showed a functional diversity of actions including antimicrobial, antiviral and cytotoxic activities (Table 2). Bioactive compounds of sponge origin have been used as basic for the synthesis of analogs, for example is glycolipids produced by bacteria that live associated with the marine spo nge Agelas sp. and the antibacterial agelasines isolated from the marine sponge Agelas nakamurai (Bakkestuen et al. 2005).

Kimura et al. (1998) had isolated 1-Methyherbipoline from Halisulfate-1 and suvanin as a serine protease inhibitor from Coscinoderma mathewsi sponge. Bioactive compounds such as macrocyclic peptide had isolated from Theonella swinhoei comes from water area at Japan. These bioactive compounds known as Cyclotheonamida A and B that have inhibitory activity to serine protease like thrombin and contains vinyl ogous tyrosine (V-Tyr) and α-ketoarginine residu which is still unknown amino acid in nature.

Table 2 Bioactive compounds produced by marine sponges (Soediro 1999; Simmons et al. 2005)

Pharmace utical

Activity Bioactive Compounds Types of Sponge

Cytotoxic 3,6 epoksieikosa acid

Swinholida A Vaskulin Halisilindramida A Jasplakinolide Jaspicamides Hymeniacidon hauraki Theonella swinhoei Cribrocalina vasculum Halichondria caveolata Jaspis johnstoni Jaspis sp.

Anticancer Agelasfin (AGL) Agelas muritianus

Anti blood cancer Kurasin A

Amfidinolid B1, B2, B3, N, Q Triangulinat acid

Lingbya majuscule Amphidinium sp. Pellina triagulata

Antiviral (HIV 1) Trikendiol Trikentrion loeve

Antimicrobial Hormotamnim

Diskodermin E-H Wondosterols

Hormothamnion Discodermia kiiensis Jaspis wondoensis

Antibacterial Lokisterolamin A and B Corticium sp.

Antifungal kortikatat acid A,B,C

Leukasandrolida Halisilindramida

Petrosia corticata Leucasandra caveolata Halichondria cylindrical

Imunomodulator Agelasflin 10 and 12 Agelas muritianus

Anti-inflammatory Manualida Luffariella variabilis

Unknown substances (still research)

Halisiklamina A Bastadin A and B Klatirimin Halisiklamina B Haliclona sp. Lanthella basta Clathria basilana Xestrospongia sp.

O’Keefe et al. (1998) had isolated Adociavirin from Adocia sp. sponge at Bay water area, New Zealand; extract that dissolved in distillation water potential as antisitopatic inside of CEM-SS cell which infection from HIV-1. Matsunaga et al. (1992) had been isolated 1-acid carboxymethylnicotinic from sponge Antosigmella raromicroscera which can be used as protease inhibitor. Li et al. (2006a) had been isolated 399 bacteria from the sponges Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in the South China Sea, among which, 13 isolates from S. tenuis, 42 from H. rugosa, and 20

from D. avara showed pronounced broad-spectrum antimicrobial activities and enzymatic potentials. Many of the pharmacologically most promising natural products from sponges are complex polyketides. The fact that polyketide synthases (PKSs) are almost absent in metazoans suggests a microbial origin. PKSs are therefore a particularly good study object to investigate the role of symbionts in the chemistry of marine sponges (Castoe et al. 2007).

Polyketide Synthas e and Nonribos omal Peptide Synthetas e

The structural characteristics of marine natural products have revealed that they mainly belong to two important chemical families, namely, polyketides and cyclopeptides, and are synthesized by multifunctional enzymes called polyketide synthases (PKSs) and nonribosomal peptide synthetase (NRPSs). Polyketides are a group of secondary metabolites, exhibiting remarkable diversity in their structures and functions. Polyketide natural prod ucts are known for their wide range of pharmacologically impor tant activities, including antimicrobial, antifungal, antiparasitic and antitumor properties (Hill 2005).

Nonribosomal peptides are part of a family of complex natural products built from simple amino acid monomers. They can be found in bacteria and fungi where they are synthesized by nonribosomal peptide synthetase (NRPS) which are large multimodular and multifunc tional proteins. Nonribosomal peptides as well as the hybrid products are of much interest because of their pharmaceutical properties such as the immunosuppressant cyclosporine (Schwartzer et al. 2003).

Schirmer et al. (2005) had already characterized PKS gene cluster in metagenomic libraries from Discordemia dissolute. The PKS gene cluster is 110 kb and contain of three open reading frames (ORF). The first PKS ORF codes for a remarkably large protein of 25.572 amino acids with a predicted molecular mass of 2.7 MDa. The most remarkable features of this large PKS gene cluster are the presence of a complete set of reductive domains (ketoreductase, dehydratase, and enoylreductase) in all except one module, which lacks the ER, and C-methyltransferase domains in 8 of the 14 modules. The products of the PKS gene clusters have more similarity with fatty acids (Figure 1).

Figure 1 Organization of the multimodular PKS gene cluster in metagenomic libraries from D. dissolute (Schirmer et al. 2005).

Nguyen (2009) had already investigated the polyketides biosynthetic pathway in the spo nge Theonella swinhoei and the beetle Paederus fuscipes. The adenylation (A) domain of an NRPS module is responsible for specific selection and activation of a defined amino acid. Expression of A domains should help us gain insights into the biosynthetic pathways of pederin and onnamides. There were two A do mains on the module PedF2 and PedH6 of the ped gene cluster and also two A domains on the module OnnI2 and OnnJ4 of the onn gene cluster. In agreement with the structure of onnamide B, the prediction results showed that glycine and arginine were the specific amino acids of NRPS modules on the onn gene cluster (Figure 2).

Figure 2 Two A domains inside the biosynthetic gene cluster of Onnamide B (Nguyen 2009).

MATERIALS AND METHODS

Duration and Place of Study

This research was carried out from July 2011 to March 2012. Extraction, fractionation and purification of antimicrobial compounds were carried out in the laboratory of Microbiology, Department of Biology and Biopharmaca Research Center, IPB, Indonesia. Molecular genetic analysis was carried out in Yohda Laboratory, Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology (TUAT), Japan. The flowchart of method s that used in this study is given in F igure 3.

Materials

Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West Papua Province by Abubakar (2009). These bacteria were used for the extraction, fractionation and purification of antimicrobial compounds. Specific primer 63 f (5-CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGGCGGWGTGTACAAG GC-3) was used for analysis of 16S rDNA (Marchesi et al. 1998). Degenerate pr imer (f: 5-GCSATGGAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTG SSCYTCSAC-3) for KS domain and degenerate primer (f: 5-AARDSIGGIGSIG SITAYBICC-3); (r: 5-CKRWAICCICKIAIYTTIAYYTG-3) for A domain (Schirmer et al. 2005).

Extraction of Antimicrobial Compounds

Each of those three isolates was sub-cultured in 500 ml Seawater Complete Broth media (bacto peptone 2.5 g, yeast extract 0.5 g, glycerol 1.5 ml, seawater 375 ml and distilled water 125 ml) and incubated in fluctuate incubator with 100 rpm at 300C until the culture reached the stationary phase. After that, 10% liquid bacterial inocula from previously incubation were cultured in 500 ml SWC broth and incubated in the same condition until reached the stationary phase for secondary metabolite production (Muller et al. 2004).

Extraction of antimicrobial compo unds was done by modifying the method from Sunaryanto et al. (2010). As much 500 ml of liquid bacterial culture were mixed with 500 ml of ethyl acetate solvent, incubated at room temperature for 24 hours and stirred for 2 hours with 250 rpm. These mixtures were separated and the ethyl acetate layers were evaporated with rotary evaporator until the drying residue was obtained as crude extract. The crude extract of bacterial cells were dissolved with ethyl acetate (pro analyze) to get 100 mg/ml concentration.

Figure 3 Flowchart of procedural steps used in this study.

Antimicrobial Activity Test

Antibacterial and antifungal activities from crude extracts were tested by using a gar diffusion method a gainst microbial test strains. As much 100 µl bacterial crude extracts dissolved in ethyl acetate (pro analyze) were applied carefully into 6 mm paper disks (Whatman) and at the same time, the disks were dried up using a

Antimicrobial Activity Test Extraction of Antimicrobial

Compounds

Fractionation of Bacterial Crude Extract

Purification of Antimicrobial Compounds Detection of Antimicrobial

Compounds

DNA Extraction

PCR Amplification of Ketos ynthase (KS) and Adenylation (A) Domain

Cloning of DNA Fragments Encod ing KS and A Domain

Sequencing and Bioinformatics Analysis of KS and A Domain

Morphological and Molecular Identification

hairdryer at 400C. After that, the disks were sterilized under UV light for 2 hours and put into agar plate that have been seeded with 1% (v/v) of microbial test strains (conc entration 1x106 CFU/ml, OD620 0.45). The plate was incubated at 40C for 3 hours to optimize the diffusion of bacterial crude extract into media. This assay was carried out in triplicate. The diameters of inhibition zones were measured in millimeters after incubation for 24 hours at 370C. Control disks were soaked with ethyl acetate solvent and prepared in the same manner (Sudirman 2010).

Detection of Antimicrobial Compounds

Antimicrobial compounds in each of the bacterial crude extract were detected using bioautography method. As much 10 µl of crude extracts were spotted on TLC plates (MERCK Silica Gel 60 F254) and eluted with vertical chromatography using n-butanol : ethyl acetate solvent mixture with the ratio of 3:7 (v/v). The spots on TLC plate were detected under UV light at 254 nm and 366 nm wave- length. After that, the retardation factor (Rf) values were calculated. The spots on TLC plate were cut off and dried up in room temperature. The developed TLC plates were sterilized under UV light for 1 hour before covered by 15 ml of melting SWC (450C) containing test strains, and incubated at 370C for 24 hours. Diameter of inhibition zone around the chromatogram indicated that the spo t was an active fraction (Sudirman 2005) .

Fractionation of Bacterial Crude Extract

Bacterial crude extract of isolate SAB E-41 was fractionated using semi automated flash chromatography (Buchi Pump Controller C-610). As much 3 g of crude extract was dissolved with chloroform- methanol solvent mixture (90%-10%) and injected into silica gel-column chromatography (column dimension 0.40 x 150 mm, particle size of silica gel 40 x 63 µm). Chloroform- methanol solvent mixture (90%-10%) was flowed into silica gel-column chromatography with the flow rate of 3.5 ml/minutes. The polarity level in the column was increased slowly by changing the methanol concentration from 20%, 30%, 50%, 70% until 90%. Two hundred and five fractions were collected (5 ml/each fraction) from fraction collector and combined into thirty fractions based on the same chromatogram.

These fractions were dried up and dissolved with chloroform- methanol-ethyl acetate solvent (1:1:1) to test their antimicrobial activity.

Purification of Antimicrobial Compounds

Fifteen active fractions were further purified using preparative thin layer chromatography (PTLC) technique. Each of the active fractions was spotted onto silica gel plate (MERCK Silica Gel 60 F254; 0.1 mm thickness) and eluted with n-butanol-ethyl acetate solvent mixture (3:7). The active spots on the silica gel plate were detected under UV light at 254 nm and 366 nm wave-length. The active spots were extracted directly from the silica gel plate and dissolved with chloroform-methanol-ethyl acetate solvent mixture (1:1:1). The active fractions that were purified by preparative TLC were tested for their antimicrobial activity.

Morpholog ical and Molecular Identification

Morphological characterization of those three isolates was performed using Gram staining p rocedure. Molecular analysis of 16S rDNA was done using specific primer, 63f (5-CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGG CGGWGTGTACAAGGC-3) (Marchesi et al. 1998). The PCR cycling condition for 16S rDNA was carried out under the following condition such as initial denaturation at 940C for 5 min, followed by 30 cycles of denaturation at 940C for 1 min, annealing at 550C for 1 min, elongation at 720C for 1 min and post PCR at 720C for 10 min. PCR products of 16S rDNA were purified using GENECLEAN® II Kit. These PCR products were sub-cloned into T-Vector pMD20 and transformed into competent E. coli DH5-α using heat shock method (Sambrook & Russell 2001). Afterwards, several steps such as PCR colony, isolation and restriction of recombinant plasmid, PCR sequencing and purification of PCR products were done before the 16S rDNA sequence analysis.

DNA Extraction

Each of bacterial isolates were sub-cultured into SWC broth media and incubated at room temperature for 24 hours. After that, 1.5 ml bacteria isolate was drawn into microtube and centrifuged (18.000xg) for 10 min to obtain bacteria

pellet that were used for DNA extraction. The supernatant was discarded and 250 µl Tris-EDTA (TE) buffer was added and centrifuged at 8000 rpm for 10 min. The supernatant was discarded and pellet was re-suspended three times in TE buffer. As much 250 µl TE and 5 µl lysozyme were added together and microtube slowly inverted to allow mixing and incubated at 370C for 30 min. After incubation, the solution was added with 500 µl SDS 10% and 10 µl proteinase K and incubated again at 370C for 60 min. Afterwards, as much 80 µl NaCl was added together with 100 µl CTAB 10% and incubated at 650C for 20 min. After incubation, added again the solution with 650 µl PCI and shake n strongl y then centrifuged at 14.000 rpm for 10 min. The upper solution was transferred into a new microtube then 650 µl CI was added and centrifuged again in same condition. DNA was precipitated using absolute ethanol (2x vol) and Na acetate 3 M 0.1 vol and incubated overnight in freezer. After that, 1 ml ethanol 70% was added for final washing and centrifuged at 12.000 rpm for 10 min. The supernatant was discarded and pellet was air dried overnight. After this step, 20 µl of TE was added and the extracted DNA was stored at -200C for further applications (Sambrook & Russel 2001).

PCR Amplification of KS and A Domain

KS domain of PKS and A do main of NRPS genes from those three isolates were amplified using PCR primers such as degenerate KS domain (f: 5-GCSATG GAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTGSSCYTCSAC-3) and degenerate A domain (f: 5-AARDSIGGIGSIGSITAYBICC-3); (r: 5-CKRWAICC ICKIAIYTTIAYYTG-3) (Schirmer et al. 2005). The PCR cycling condition for KS domain was carried out in three steps such as initial denaturation at 940C for 5 min, followed by 35 cycles of denaturation at 940C for 1 min, annealing at 500C for 1 min, elongation at 720C for 1 min 10 sec and post PCR at 720C for 10 min. The PCR cycling condition for A domain was the same as for KS domain except for annealing which was carried out at 550

In all cases, the reaction mixtures contained 4 µl dNTP mix (2.5 mM), 5 µl 10X Ex Taq buffer, primer forward and reverse (10 µM; each of 5 µl), 2 µl DNA template (500 ng/µl), 1 µl TaKaRa Ex Taq

C for 1 min.

TM

total volume of reaction mixtures was 50 µl. PCR products were analyzed using agarose gel electrophoresis 1% (b/v).

Cloning o f DNA Frag me nts Encoding KS and A Domain

Purification of PCR products from KS domain of PKS gene (700 bp) and A domain of NRPS gene (1000 bp) were carried out using GEN ECLEAN® II Kit. Each of the purification prod ucts was sub-cloned into T-Vector pMD20 (TaKaRa) and transformed into competent E. coli DH5α using heat shock method (Sambrook & Russell 2001). Isolation of recombinant plasmid was performed using Mag ExtractorT M Quick Plasmid Miniprep kit (Toyobo, Japan). Restriction of recombinant plasmid was conducted using the combination of restriction enzymes such as BamHI (BioLabs) and XbaI (TaKaRa). The reaction mixtures contained 0.2 µl BamHI (5 units/µl), 0.2 µl XbaI (5 units/ µl), 1 µl 10X NE buffer 4, 0.5 µl DNA template (500 ng/µl) and 8.1 µl milli Q. The total volume of reaction mixtures was 10 µl. The reaction mixtures were incubated at 370C for 24 hours and the restriction product was analyzed using agarose gel electrophoresis 1% (b/v).

Sequencing a nd Bioinformatics Analysis of KS and A Domain

DNA fragments that were inserted into plasmid T-Vector pMD20 named pMD20-KS domain and pMD20-A domain were used for the sequencing process. M13 primer RV and M13 primer M4 were used for PCR sequencing. The PCR cycling condition was carried in three steps such as initial denaturation at 960C for 5 min, followed by 25 cycles of denaturation at 960C for 1 min, annealing at 500C for 30 sec, elongation at 600C for 1 min and post PCR at 40C for an unlimited time. The Big Dye® X TerminatorT M purification kit was used to purify the PCR product before DNA sequencing. The DNA was run in an automated DNA sequencer using ABI 3130 XL Genetic Analyzer. The DNA sequences were compared to the database available at NCBI using BlastN program for 16S rDNA and BlastX program for KS and A domain. Construction of phylogenetic tree was carried out using MEGA5 program with neighbor-joining method.

RESULTS

Antimicrobial Activity o f Bacterial Crude Extracts

Antimicrobial compounds of three bacterial isolates can be extracted using ethyl acetate solvent. During the extraction process, the ethyl acetate solvent will be formed two layers. First layer was aqueous phase that contained the antimicrobial compounds which have been extracted by ethyl acetate solvent. Second layer was organic phase that contained the bacterial cultures. The first layer was obtained and separated from bacterial cultures then evaporated with rotary evaporator until the drying residue was obtained as crude extract.

Crude extracts of isolates SAB E-31, SAB E-41 and SAB E-57 showed different antimicrobial activity against non-pathogenic and pathogenic microbes. The bacterial crude extract of isolate SAB E-41 showed better antimicrobial activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57. Crude extracts of those three isolates demonstrated the best activity against S. aureus (Table 3).

Table 3 Diameter average of inhibition zone (mm) from three bacterial crude extracts (100 mg/ml) produced by sponge-associated bacteria

Bacte rial Isolates Diame te r Ave rage of Inhibition Zone (mm)

BS* SA** EC* PA** EPEC K1-1** CA** CT**

SAB E-31 8.3 10.5 5.3 4.8 5.6 2.5 2.5

SAB E-41 10.5 14.5 7.5 6.7 8.7 5.7 4.5

SAB E-57 9.5 13.2 6.4 5.8 6.5 3.8 2.8

Positive Control (Ampicilin 100 mg/ml)

Negative Control

14 30 9 27 - - -

(Ethyl acetate) - - - -

BS = B. subtilis; SA = S. aureus; EC = E. coli; PA = P. aeruginosa; EPEC K1-1 = Enteropa thogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; * = non-pathogen; ** = pa thogen.

Crude extracts of those three isolates showed different antimicrobial activity against EPEC K1-1, C. albicans and C. tropicalis whereas for positive control with ampicilin 100 mg/ml, there was no inhibition a gainst these pathogenic microbe s (Figure 4). Ethyl acetate solvent that used as a negative control didn’t inhibit the growth of non-pathogenic or pathogenic microbes.

Figure 4 Antimicrobial activity of three bacterial crude extracts isolates SAB E-31, SAB E-41 and SAB E-57 using agar diffusion method; C+ = positive control (ampicilin 100 mg/ml); C- = negative control (ethyl acetate).

Thin Layer Chromatography and Bioautography

TLC analysis for three bacterial crude extracts showed that there were many spots in silica gel plate with many k inds of retardation factor (Rf) values (Table 4). The solvent system, n-butanol-ethyl acetate with the ratio of 3:7 had successfully separated the component of bacterial crude extract. Six spots and many kinds of Rf values were obtained from this solvent system (Figure 5).

Table 4 Variation of Rf values from three bacterial crude extracts eluted with different solvent systems

Solve nt Syste ms Crude Extracts

Numbe r of Spots Rf Value s

λ 254

nm

λ 366 nm

λ 254

nm

λ 366

nm

n-but : CH3COOH :

ddH2 (3:1:1) O SAB E-31 SAB E-41 SAB E-57 3 3 3 2 2 2

0.80; 0.61; 0.31 0.91; 0.72; 0.35 0.88; 0.72; 0.35

0.80; 0.61 0.91; 0.72 0.88; 0.72 n-but : EtOAc :

ddH2 (2:3:1) O SAB E-31 SAB E-41 SAB E-57 3 4 4 2 2 2

0.91; 0.53; 0.44 0.91; 0.83; 0.67; 0.55 0.92; 0.85; 0.61; 0.50

0.91; 0.53 0.91; 0.83 0.92; 0.85

n-but : EtOAc (3:7) SAB E-31 SAB E-41 SAB E-57 6 6 6 3 3 3

0.88; 0.72; 0.62; 0.55; 0.44; 0.23 0.92; 0.77; 0.66;

0.60; 0.46; 0.27 0.91; 0.75; 0.65;

0.56; 0.45; 0.31

0.88; 0.55; 0.23 0.92; 0.60; 0.27 0.91; 0.56; 0.31

Figure 5 Profile of each bacterial crude extract on silica gel plate merck 60 F254 eluted with n-butanol and ethyl acetate mixture (3:7); Red box: active spots/fractions.

The spots/fractions which were detected under UV light at 254 and 366 nm wave- length were further tested for their antimicrobial activity using bioautography method. This method was able to quickly detect which spots/fractions were the active fractions or pollutant compounds in bacterial crude extract. Part of the silica gel plate was cut based on the separated spots/fractions and the different of Rf values in order to make the visualization of inhibition zones clearer. Diameter of inhibition zone around the chromatogram indicated that the spot was an active fraction (Figure 6). Bioautography detection resulted that at least 4 active spots/fractions showed antimicrobial activity against P. aeruginosa and 2 active spots/fractions which inhibited the growth of S. aureus (Table 5).

Figure 6 Antimicrobial activity of active spo ts/fractions using bioautography method.

Table 5 Active spots/fractions of three bacterial crude extracts detected using bioautography method

Bacte rial

Isolates Rf Value s

Microbial Test Strains

SA** PA** CA**

SAB E-31

Rf1 = 0.88 - - -

Rf2 = 0.72 - - -

Rf3 = 0.62 - + -

Rf4 = 0.55 - ++ -

Rf5 = 0.44 - ++ -

Rf6 = 0.23 - ++ -

SAB E-41

Rf1 = 0.92 - - -

Rf2 = 0.77 +++ - -

Rf3 = 0.66 +++ ++ -

Rf4 = 0.60 - +++ -

Rf5 = 0.46 - +++ -

Rf6 = 0.27 - ++ -

SAB E-57

Rf1 = 0.91 - - -

Rf2 = 0.75 +++ - -

Rf3 = 0.65 - ++ -

Rf4 = 0.56 + +++ -

Rf5 = 0.45 - +++ -

Rf6 = 0.31 - ++ -

+ = Weak inhibition; ++ = Medium inhibition; +++ = Strong inhibition; SA = S. aureus; PA = P. aeruginosa; CA = C. albicans; ** = pathogen.

Fractionation of Bacterial Crude Extract from Isolate SAB E-41

Two hundred and five fractions were successfully collected from the fractionation process and combined into thirty composite fractions based on the same chromatogram. These composite fractions were evaporated until the drying residue was obtained as crude extract. These crude extracts were dissolved with chloroform- methanol-ethyl acetate mixture (1:1:1) in concentration of 100 mg/ml. Antimicrobial activity of thirty compos ite fractions were tested to S. aureus, P. aeruginosa, EPEC K1-1, C. albicans and C. tropicalis.

Fifteen composite fractions coded as 1, 2, 3, 4, 5, BA-6, BA-7, BA-8, BA-11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 showed different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and C. albicans (Table 6). Fraction BA-2 that was eluted by chloroform- methanol (90%-10%) solvent system showed antifunga l activity against C. albicans whereas no active fractions showed antimicrobial activity against C. tropicalis.

Table 6 Antimicrobial activity of thirty composite fractions collected from silica gel-column chromatography

Solve nt Syste ms and Fractions Diame te r of Inhibition zone (mm) SA** PA** EPEC K1-1** CA** CT** Chloroform-Methanol (90%-10%) Fraction BA-1 Fraction BA-2 Fraction BA-3 Fraction BA-4 Fraction BA-5 Fraction BA-6 Fraction BA-7 7 8 7 3 5 4 4 2 6 4 3 4 4 2 7 10 3 10 3 2 3 - 3 - - - - - - - - - - - - Chloroform-Methanol (80%-20%) Fraction BA-8 Fraction BA-9 Fraction BA-10 2 - - - - - - - - - - - - - - Chloroform-Methanol (70%-30%) Fraction BA-11 Fraction BA-12 3 10 3 3 - - - - - - Chloroform-Methanol (50%-50%) Fraction BA-13 Fraction BA-14 Fraction BA-15 Fraction BA-16 Fraction BA-17 Fraction BA-18 14 2 2 - 12 3 2 - - - - - 7 - - - - - - - - - - - - - - - - - Chloroform-Methanol (30%-70%) Fraction BA-19 Fraction BA-20 Fraction BA-21 Fraction BA-22 Fraction BA-23 - - - - - - - - - - - - - - - - - - - - - - - - - Chloroform-Methanol (20%-80%) Fraction BA-24 Fraction BA-25 Fraction BA-26 Fraction BA-27 - - - - - - - - - - - - - - - - - - - - Chloroform-Methanol (10%-90%) Fraction BA-28 Fraction BA-29 Fraction BA-30 - - - - - - - - - - - - - - -

Negative Control (Chloroform-Methanol) - - - - -

Positive Control (Ampicilin 100 mg/ml) 30 27 - - -

SA = S. aureus; PA = P. aeruginosa; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; ** = pathogen.

Fraction BA-13 that was eluted by chloroform- methanol (50% -50%) solvent system demonstrated the highest inhibition against S. aureus followed by fraction BA-17. The diameter of inhibition zone that formed by these two active

fractions were about 12 mm and 14 mm (Figure 7). Fraction BA-2 and BA-4 that was eluted by chloroform- methanol (90% -10%) showed the best activity against EPEC K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm (Figure 7).

Figure 7 Antimicrobial activity of thirty compos ite fractions; C+ = positive control (ampicilin 100 mg/ml); C- = negative control (chloroform- methanol).

Purificat ion of Antimicrobial Compounds using Preparative TLC

Fifteen composite fractions that had an active fraction were further purified using PTLC technique. Antimicrobial compounds in active fractions could be extracted directly from silica gel plate and dissolved with chloroform- methanol-ethyl acetate (1:1:1). Antimicrobial compounds of active fractions, obtained from PTLC technique were tested to S. aureus, EPEC K1-1 and C. albicans.

Fraction BA-13 has demonstrated as the most antimicrobial compounds compared to the other fractions (Table 7). This fraction resulted four different of active compounds with Rf values of 0.87; 0.50; 0.41 and 0.12 (Figure 8). These four active compounds displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with Rf values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18 (Figure 8). Both of these active compounds showed activity against S. aureus. Fraction BA-2 carried one active compound (Rf 0.77) that showed activity against C. albicans (Figure 9).

Table 7 Rf values of active compounds from fifteen composite fractions

Active

Compounds Rf Value s

Diame te r of Inhibition Zone (mm)

SA** EPEC K1-1** CA**

BA-1 Rf1 = 0.81 4 4 -

BA-2 Rf1 = 0.77 8 14 2

BA-3 Rf1 = 0.78 2 2 -

BA-4 Rf1

Rf

= 0.87

2

6

= 0.66 -

4 -

- -

BA-5 Rf1

Rf

= 0.81

2

10

= 0.53 -

2 -

- -

BA-6 Rf1

Rf = 0.87 2 Rf = 0.65 3 10 = 0.35 - - 2 - - - - -

BA-7 Rf1

Rf = 0.87 2 Rf = 0.62 3 6 = 0.38 - - 4 - - - - -

BA-8 Rf1

Rf = 0.90 2 Rf = 0.71 3 8 = 0.35 - - - - - - - -

BA-11 Rf1

Rf

= 0.87

2

3

= 0.68 -

- -

- -

BA-12 Rf1

Rf = 0.90 2 Rf = 0.71 3 Rf = 0.62 4 Rf = 0.41 5 10 = 0.12 - - - - - - - - - - - - - -

BA-13 Rf1

Rf = 0.87 2 Rf = 0.72 3 Rf = 0.60 4 Rf = 0.50 5 Rf = 0.41 6 3 = 0.12 - - 4 8 12 10 - - 6 4 2 - - - - - -

BA-14 Rf1

Rf = 0.90 2 Rf = 0.75 3 Rf = 0.68 4 2 = 0.58 - - - - - - - - - - -

BA-15 Rf1

Rf = 0.93 2 Rf = 0.78 3 Rf = 0.68 4 2 = 0.33 - - - - - - - - - - -

BA-17 Rf1

Rf = 0.93 2 Rf = 0.71 3 Rf = 0.68 4 Rf = 0.33 5 14 = 0.12 - - - 2 - - - - - - - - - -

BA-18 Rf1

Rf = 0.93 2 Rf = 0.41 3 Rf = 0.25 4 2 = 0.12 - 2 - - - - - - - - -

SA = S. aureus; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; ** = pathogen.

Figure 8 Profile of active compounds from fraction BA-13, BA-17 and BA-18 on silica gel plate merck 60 F254 (Arrow head).

Figure 9 Antimicrobial activity of fifteen compos ite fractions purified using PTLC technique; C- = negative control (ethyl acetate).

Morphological and Molecular Identification Bas ed on 16S rDNA Analysis Morphological characterization of isolates SAB E-31, SAB E-41 and SAB E-57 was carried out using Gram staining bacteria. Gram staining results showed that three marine bacterial isolates were rod-shaped, motile, formed spores and Gram-pos itive bacteria. These isolates have the same characteristics with the genus of Bacillus (Figure 10).

Figure 10 Gram staining of three bacterial isolates coded as: A). SAB E-31; B). SAB E-41 and C). SAB E-57.

BA-13 BA-17 BA-18

Active Co mpounds (Rf 0.87)

Active Co mpounds (Rf 0.50) Active Co mpounds

(Rf 0.41)

Active Co mpounds (Rf 0.12)

Active Co mpounds

(Rf 0.93)

Active Co mpounds

(Rf 0.12)

Active Co mpounds

(Rf 0.93)

Active Co mpounds

(Rf 0.25)

λ 366 nm λ 366 nm λ 254 nm

A. _{2 µm} B. _{2 µm} C.

Molecular analysis of 16S rDNA was carried out for the further identification of those three isolates. PCR product of 16S rDNA sequences was about 1300 bp. Sequences analysis of 16S rDNA showed a high similarity level (97-98%) to various strains of Bacillus compared to those available in GenBank database (Table 8).

Table 8 Similarity of 16S rDNA sequences from isolates SAB E-31, SAB E-41 and SAB E-57 compared with GenBank Database

Bacte rial

Isolates Similarity

Ide ntity (%)

E-

Value Accession

SAB E-31 Bacillus pumilus strain KD3 98 0.0 EU500930.1

SAB E-41 Bacillus amyloliquefaciens strain zy2 98 0.0 JN160740.1

SAB E-57 Bacillus subtilis strain YRL02 97 0.0 EU373407.1

Phylogenetic analysis of 16S rDNA sequences showed that isolates SAB E-31, SAB E-41 and SAB E-57 formed a different clade with the reference strains of Bacillus in GenBank database. Clade-1 was formed by the reference strains of Bacillus while clade-2 was formed by those three isolates with Bacillus sp. DF49 (Figure 11).

Figure 11 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains based on 16S rDNA sequences. Numbers at the node s indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.1 substitutions per nucleotide pos ition.

Amplification of DNA Frag me nts Encoding KS and A Domai n

DNA fragments of three bacterial isolates encoding KS and A domain were successfully amplified using PCR. Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had a DNA fragment encoding A do main whereas only two bacterial isolates coded as SAB E-41 and SAB E-57 had a DNA fragment encoding KS do main. Visualization of the PCR amplicon for DNA fragments was analyzed using agarose gel electrophoresis 1% (b/v). DNA fragment encoding KS domain was in a size of 700 bp while for A domain was in a size of 1000 bp (Figure 12).

Figure 12 Agarose gel electrop horesis of DNA fragments encod ing KS Domain and A Domain.

Cloning a nd Bioinformatics Analys is

DNA fragments of those three isolates encoding KS and A domain were successfully cloned into T-Vector pMD20 (TaKaRa Bio Inc.) and named pMD20-KS domain and pMD20-A domain. White colonies of E. coli DH5-α carrying the recombinant plasmid had been isolated and for the plasmid excision was do ne using a combination of restriction enzymes, BamHI and XbaI. The cropped recombinant plasmid DNA resulted in two bands which were approximately of 2736 bp that show ed the size of T- vector pMD20 and 700 bp was the size of DNA fragment encoding KS domain and 1000 bp for DNA fragment encoding A do main (Figure 13).

1 k b L a d d e r S AB E -3 1 S AB E -3 1 S AB E -4 1 S AB E -5 7 S AB E -4 1 S AB E -5 7 1 kb 250 500 750

1000 _{A Do main (~1000 bp)}

KS Do ma in (~700 bp) bp

A B

Figure 13 Restriction of recombinant plasmids digested with BamHI + XbaI; A). pMD20-KS domain and B). pMD20-A do main.

Bioinformatics sequences analysis of DNA fragment encoding KS domain from two bacterial isolates coded as SAB E-41 and SAB E-57 using BlastX program showed that isolate SAB E-41 had a similarity level of 97 % with type I PKS from Bacillus amyloliquefaciens LL3 while isolate SAB E-57 had a similarity level of 98% with putative polyketide synthase pksL from B. amyloliquefaciens subsp. plantarum CAU-B946 (Table 9).

Table 9 Bioinformatics sequences analysis of DNA fragment encoding KS domain using BlastX program

Bacte rial

Isolates Similarity

Ide ntity (%)

E-

Value Accession

SAB E-41 Type I PKS; B.

amyloliquefaciens LL3

97 2e-130 YP_005545643.1

SAB E-57 Putative polyketide synthase

pksL; B. amyloliquefaciens

subsp. plantarum CAU-B946

98 2e-143 YP_005130937.1

Sequences analysis of DNA fragment encoding A domain showed that isolate SAB E-31 had a similarity level of 81% with bacitracin synthetase 1 from B. pumilus ATCC 7061. Isolate SAB E-41 had a similarity level of 80% with surfactin 19329 7743 6223 4254 3472 2690 1882 1489 925 421 19329 7743 6223 4254 3472 2690 1882 1489 925 421

A Do main (~1000 bp) T-Vector pMD20 (2736 bp) T-Vector pMD20 (2736 bp)

KS Do ma in (~700 bp) λ M ar ke r C o n tr o l S AB E -4 1 S AB E -5 7 λ M ar ke r C o n tr o l S AB E -4 1 S AB E -3 1 S AB E -5 7

synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 while isolate SAB E-57 had a similarity level of 81% with surfactin synthetase A from B. amyloliquefaciens subsp. plantarum CAU-B946 (Table 10).

Table 10 Bioinformatics sequences analysis of DNA fragment encoding A domain using BlastX program

Bacte rial

Isolates Similarity

Ide ntity (%)

E-

Value Accession

SAB E-31 Bacitracin synthetase 1; B.

pumilus ATCC 7061

81 1e-154 ZP_03054623.1

SAB E-41 Surfactin synthetase B; B.

amyloliquefaciens subsp. plantarum CAU-B946

80 1e-139 YP_005129035.1

SAB E-57 Surfactin synthetase A; B.

amyloliquefaciens subsp. plantarum CAU-B946

81 1e-129 YP_005129034.1

Amino acid sequences of KS domain from isolates SAB 41 and SAB E-57 were aligned and compared with the reference strains in GenBank database. Two bacterial isolates have a homology of conserved region with the reference strains (Figure 14). Phylogenetic analysis of amino acid sequences of KS domain showed that isolates SAB E-41 and SAB E-57 formed a different clade with the other reference strains (Figure 15).

Figure 14 Alignment of amino acid sequences of KS domain from isolates SAB E-41 and SAB E-57 with the reference strains in GenBank Database using ClustalW program. Shaded area showed the similarity of amino acid sequences. Black bo x showed a homology of conserved region.

Figure 15 Phylogenetic tree of isolates SAB E-41 and S AB E-57 with the reference strains based on amino acid sequences of KS domain. Numbers at the nodes indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.1 substitutions per nucleotide pos ition.

Clade-1

Amino acid sequences of A domain from isolates SAB E-31, SAB E-41 and SAB E-57 were also aligned and compared with the reference strains in GenBank database. These bacteria have a homology of conserved region with the other reference strains (Figure 16). Phylogenetic analysis of amino acid sequences of A domain showed that isolate SAB E-41 was formed a similar clade with B. amyloliquefaciens FZB42 while isolates SAB E-31 and SAB E-57 formed a same clade (Figure 17).

Figure 16 Alignment of amino acid sequences of A domain from isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains in GenBank Database using ClustalW Program. Black box showed a homology of conserved region for amino acid sequences.

Figure 17 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains based on amino acid sequences of A domain. Numbers at the nodes indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.2 substitutions per nucleotide pos ition.

DISCUSSION

Antimicrobial compounds of three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 can be directly extracted using ethyl acetate solvent. The selection of this solvent system for the extraction process is based on the polarity difference of the liquid culture of bacteria. This solvent is non-polar so that the separation of liquid culture of bacteria from ethyl acetate layer was more easily done. Jeffery et al. (1989) stated that antimicrobial compounds contained in bacterial crude extract have different solubility prope rties in each of solvent system. The selection of an appropriate solvent in the extraction process is largely determined by the solubility properties of the antimicrobial compounds, type of substrate, the partition coefficient and the distribution ratio of solvent system.

Crude extracts of those three isolates showed different antimicrobial activity against non-pathogenic and pathogenic microbes. The highest activity shown by the bacterial crude extracts of isolate SAB E-41 which indicated from the large diameter of the inhibition zone. Lay (1994) stated that physical and chemical properties of antimicrobial compounds will affect the resulting of clear zone. The greater of the molecular weight of bioactive compounds, the more enlarge of the inhibition zone. The other factors that also affecting the inhibition zone are the density of cells, the sensitivity of microbial test strains to antimicrobial compounds, the component of antimicrobial compounds, the diffusion rate of antimicrobial compounds into media and the expos ure time of microbial test strains to antimicrobial compounds.

On the ot her hand, crude extracts of those three isolates showed the highest activity against S. aureus whereas the lowest activity shown by the test strains of C. albicans and C. tropicalis. The antimicrobial compounds from three bacterial crude extracts couldn’t optimally inhibit the growth of pa thogenic fungal because of the lower concentration. For the comparison in tested their antimicrobial activity, ampicillin was used as a positive control whereas ethyl acetate solvent was used as a negative control. Ampicillin which included in - lactam group of antibiotics had a broad spectrum activity so it was chos e as a positive control for this study. This ant ibiotic unable to inhibit the growth of enteropathogenic E. coli K1-1 because of

the - lactamase enzyme that prod uced by this microbe while interestingly, our crude extracts from three bacterial isolates which associated with sponge Jaspis sp. are able to inhibit the growth of this microbe as seen from the diameter average of inhibition zone.

Anand et al. (2006) reported that antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India which guided fractionation of the broth showed that the ethyl acetate extract of strain SC3 demonstrated activity against the bacterial and fungal test strains. The strain SC3 showed the highest activity against test strains of B. subtilis and E. coli with an inhibition zone of 26 mm and for C. albicans of 15 mm.

TLC analysis and bioautography test were performed for three bacterial crude extracts. The TLC analysis aims to separate the constituent components of bacterial crude extract based on the difference of absorption, partition and solubility of the chemical components that will be moved with the polarity of eluent whereas bioautography test aims to quickly detect which spots were the active compounds or impurities compounds. Detection of the spots was performed by administering the chromatogram plates under UV light with 254 nm and 366 nm wave- length. All of the detection methods that used in this study are expected to quickly detect the presence of active compo unds without needed to detect the spo ts one by one on silica gel plate. In this study, detection of active compounds was only performed with these methods so that not all patches of the active compounds contained in bacterial crude extract can be detected.

Sudirman (2005) reported that the rapid detection of active compounds contained in bacterial crude extract can be done using bioautography method besides the UV irradiation and the spraying of color-forming reaction. Bioautography technique is a combination of chemical methods (chromatography) with the microbiological method, the chromatogram plates covered with media that contained microbial test strains and incubated according to the growth temperature of test strains. Betina (1964) stated that bioautography method can be directly detected the activity and the minimum number of active compounds that contained in the bacterial crude extract. In addition, this method can be directly detected the

specify location of active compounds based on the position or the Rf values on the chromatogram plate.

Four active spots/fractions with the Rf values of 0.62; 0.55; 0.44; 0.23 (isolate SAB E-31), Rf 0.66; 0.60; 0.46; 0.27 (isolate SAB E-41) and Rf 0.65; 0.56; 0.45; 0.31 (isolate SAB E-57) were successfully detected using these methods and respectively showed antimicrobial activity against P. aeruginosa, while two active spots/fractions with the Rf values of 0.77; 0.66 (isolate SAB E-41) and Rf 0.75; 0.56 (isolate SAB E-57) respectively displayed antimicrobial activity against S. aureus. No active spots/fractions from isolates SAB E-31 can inhibit the growth of S. aureus.

Banoet (2011) reported that at least one active spot/fraction was successfully detected using TLC analysis and bioautography detection. Two spots/fractions with the Rf value of 0.31; 0.81 from ethyl acetate extract of isolate HAL-13 and one spot/fraction with the Rf value of 0.85 from n-butanol extract of isolate HAA-01 and Rf 0.28 from the same extract of isolate HAL-74 displayed antimicrobial activity against S. aureus and enterop athogenic E. coli K1-1.

Bacterial crude extract of isolate SAB E-41 was further fractionated using column chromatography techniques. The selection of this crude extract for further testing due to the be st activity against non-pathogenic and pathogenic microbes compared to the other bacterial crude extracts. Purification of antimicrobial compounds via column chromatography techniques is based on the polarity of eluent. Crude extract was slowly injected into the silica gel-column chromatography and this extract will be separated based on the difference of the polarity of eluent system that used d uring the elucidation process.

Hurtubise (2010) mentioned that chromatography column was a classic chromatography method which used to separate the constituent components of antimicrobial compounds for the large quantities by adsorption and partition mechanism. The principles of this process were based on the different polarity of active compounds that contained in the bacterial crude extract. The right selection of stationary phase (absorbance) and mobile phase (eluent) in fractionation process will be determined the successful of the separation of crude extract. The solvent was allowed to flow through the column due to the gravity or the pressure. The

Appendix 9 Alignm ent of DNA sequences encoding KS domain of PKS gene from isolate SAB E-57 using BlastX program

amyloliquefaciens subsp. plantarum CAU B946]

amyloliquefaciens subsp. plantarum CAU B946]

Length=2071

[Bacillus amyloliquefaciens CAU-B946]

Score = 451 bits (1160), Expect = 2e-143

Identities = 218/222 (98%), Positives = 219/222 (99%) Gaps = 0/222 (0%), Frame = -2

Query 666 DPQQRVFLEESWKALGDAGYAGDSVRGRECGVYAGSCGGDYQTIFKQQGPAQAFWGNHNS 487 DPQQR+FLEESWKAL DAGYAGDSVRGRECGVYAGSCGGDYQ IFKQQGPAQAFWGNHNS Sbjct 954 DPQQRLFLEESWKALEDAGYAGDSVRGRECGVYAGSCGGDYQAIFKQQGPAQAFWGNHNS 1013 Query 486 VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ 307 VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ Sbjct 1014 VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ 1073 Query 306 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD 127 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD Sbjct 1074 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD 1133 Query 126 GATNGITAPSALSQERLERHVYDTFHINPETIQMVEGHGTGT 1

GATNGITAPSALSQERLERHVYDTFHINPETIQMVE HGTGT Sbjct 1134 GATNGITAPSALSQERLERHVYDTFHINPETIQMVEAHGTGT 1175

Appendix 10 Alignment of DNA sequences encoding A domain of NRPS gene from isolate SAB E-31 using BlastX program

bacitracin synthetase 1 (BA1) [Bacillus pumilus

ATCC 7061]

7061]

Length=3570

Score = 492 bits (1266), Expect = 1e-154

Identities = 263/323 (81%), Positives = 281/323 (87%) Gaps = 6/323 (2%), Frame = -1

Query 969 DERIQGTF*QDSGAQFVPDPIQVLRHRSVLTAFEGGKS*KQKIQAVDQQSESNPSLYVFR 790 DER++ F DSGAQF+ QVLRHRSVL +FEG + + + + QQS+SN + V Sbjct 1588 DERVKH-FLTDSGAQFLLTH-QVLRHRSVLASFEGTII-ETEDRGIVQQSDSNIDIRVLP 1644 Query 789 LWDLGEF*PTTCWYDRQNLKGNMVTHRNILRTVKQSNYLTIHHEDTVMSLSNYVFDAFMF 610 DL T+ + KGNMVTHRNILRTVKQSNYL IHHEDTVMSLSNYVFDAFMF Sbjct 1645 E-DLANLTYTSGTTGKP--KGNMVTHRNILRTVKQSNYLAIHHEDTVMSLSNYVFDAFMF 1701 Query 609 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKGSLKNVR 430 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKK SLKNVR Sbjct 1702 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKDSLKNVR 1761 Query 429 KVLFGGERASVPHVVAALETVGEDKLIHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV 250 KVLFGGERASVPHV+ ALETVGE KL+HMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV Sbjct 1762 KVLFGGERASVPHVMTALETVGEGKLVHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV 1821 Query 249 SQTAVYIVDEFGHVQPPGVAGELCVAGDGLVKGYYRQPELTSEEFVENPFRPGEVMYKTG 70 SQTAVYIVDEFG +QPPGVAGELCVAGDGLVKGYY QP+LTSE+FVENPFRPGEVMYKTG Sbjct 1822 SQTAVYIVDEFGQLQPPGVAGELCVAGDGLVKGYYGQPKLTSEKFVENPFRPGEVMYKTG 1881 Query 69 DLARWLSNGDIEFIGRIDHQVKI 1

DLARWLSNG+IEFIGRIDHQVKI Sbjct 1882 DLARWLSNGEIEFIGRIDHQVKI 1904

Appendix 11 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-41 using BlastX program

amyloliquefaciens subsp. plantarum CAU B946]

subsp. plantarum CAU B946]

Length=3586

[Bacillus amyloliquefaciens CAU-B946]

Score = 449 bits (1154), Expect = 1e-139

Identities = 236/295 (80%), Positives = 246/295 (83%) Gaps = 6/295 (2%), Frame = +1

Query 112 KEQAGTLQVPIVMLDEKRG*--NGKRNRLESSGRRATTWRISCIHPDRPANRKAS*LNHR 285 +EQAGTLQVPIVMLDE +G L + G + +P K + HR Sbjct 573 QEQAGTLQVPIVMLDESADETVSGTDLNLPAGGNDLAYIMYTSGSTGKP---KGVMIEHR 629 Query 286 NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA 465 NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA Sbjct 630 NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA 689 Query 466 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW 645 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW Sbjct 690 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW 749 Query 646 NGYGPTENTTFSTSFLIDQDCDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG 825 NGYGPTENTTFSTSFLIDQD DGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG Sbjct 750 NGYGPTENTTFSTSFLIDQDYDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG 809 Query 826 VARGYVNLPELTEKQFVRDPFRPEKRYTRTGDLAKDGFPAARTSFLAEMATQEKI 990 VARGYVNLPELTEKQFVRDPFRP++ RTGDLAK P FL + Q K+ Sbjct 810 VARGYVNLPELTEKQFVRDPFRPDETIYRTGDLAK-WLPDGTIEFLGRIDNQVKV 863

Appendix 12 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-57 using BlastX program

amyloliquefaciens subsp. plantarum CAU B946]

amyloliquefaciens subsp. plantarum CAU B946]

Length=3584

[Bacillus amyloliquefaciens CAU-B946]

Score = 420 bits (1080), Expect = 1e-129

Identities = 227/280 (81%), Positives = 236/280 (84%) Gaps = 15/280 (5%), Frame = -1

Query 845 NNKSDRAWLYIIYTSgnngggrRA**LSTGPNVHHLVQSLQQEIYQCGEQTLRMALLAPL 666 + +SDR YIIYTSG G + + VHHLVQSLQQEIYQCGEQTLRMALLAP Sbjct 593 STQSDRL-AYIIYTSGTTGRPKGV--MIEHRQVHHLVQSLQQEIYQCGEQTLRMALLAPF 649 Query 665 HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD 486 HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD Sbjct 650 HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD 709 Query 485 VSGIELRHMLIGGEGLSAAVAEQLMNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG 306 VSGIELRHMLIGGEGLSAAVAEQL+NLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG Sbjct 710 VSGIELRHMLIGGEGLSAAVAEQLLNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG 769 Query 305 MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLSGFDPQRSF 126 MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNL ++ F Sbjct 770 MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLPDLTAEK-F 828 Query 125 LQDPFNGSGRYVPHRVIWRAG---CRTGRSNIFGREDD 21

LQDPFNGSGR ++R G G GREDD Sbjct 829 LQDPFNGSGR---MYRTGDMARWLPDGTIEYIGREDD 862

SUMMARY

EFFENDI. Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is. Under direction of ARIS TRI WAHYUDI and MUNTI YUHANA.

The increase of global resistance of the pathogenic microbes against various antibiotics becomes a serious concern in public health. Many efforts are conducted in order to solve this problem, such as finding the new bioactive compounds from marine bacteria which associated with marine sponges. Three marine bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that associated with sponge

Jaspis sp. showed their capability in producing antimicrobial compounds. In this

study, extraction of antimicrobial compounds from these bacteria was conducted using ethyl acetate solvent. Each of bacterial crude extract displayed significant activity against B. subtilis, E. coli as non-pathogenic microbes and S. aureus, P.

aeruginosa, enterop athogenic E. coli K1-1, C. albicans and C. tropicalis as

pathogenic microbes. Bacterial crude extract of isolate SAB E-41 demonstrated better activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57.

Analysis of constituent component for each bacterial crude extract was conducted using thin layer chromatography (TLC). Each of bacterial crude extract was spotted onto silica gel plate and eluted with n-butanol-ethyl acetate solvent mixture (3:7). Six spots/fractions were successfully detected by viewing under UV light at 254 nm and 366 nm wave- length. Active spots/fractions from each bacterial crude extract were detected using bioautography method. Four spots from each bacterial crude extract showed antimicrobial activity against P. aeruginosa and t wo spots showed antimicrobial activity against S. aureus.

Fractionation of bacterial crude extract from isolate SAB E-41 was carried out using silica gel-column chromatography. Two hundred and five fractions were successfully collected from fraction collector and combined into thirty compos ite fractions based on the same chromatogram by using TLC ana lysis. The antimicrobial activity of thirty compos ite fractions was tested to S. aureus, P.

aeruginosa, enteropathogenic E. coli K1-1, C. albicans and C. tropicalis. F ifteen of

the m named by 1, 2, 3, 4, 5, 6, 7, 8, 11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 have different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and C. albicans. Fraction BA-2 that was eluted by chloroform- methanol (90% -10%) solvent system showed antifungal activity against C. albicans while fraction BA-13 that was eluted with chloroform-methanol (50% -50%) solvent system showed the highest inhibition against S.

aureus followed by fraction BA-17. The diameter of inhibition zone that formed by

these two active fractions were about 12 mm and 14 mm. Fraction BA-2 and BA-4 that was eluted with chloroform- methanol (90%-10%) showed the best activity against enteropathogenic E. coli K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm.

Purification of fifteen compos ite fractions was conducted using preparative thin layer chromatography (PTLC) technique. Four active fractions with the Rf values of 0.87; 0.50; 0.41 and 0.12 were successfully collected from fraction

BA-13 and displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with the Rf values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18. Bot h of these active compounds were successfully collected and showed antimicrobial activity against S.

Morphological and molecular identification of those three isolates were carried out in order to identify these bacteria. Sequences analysis of 16S rDNA showed that three isolates were included in the genus of Bacillus. Isolate SAB E-31 had 98% of homology level with B. pumilus strain KD3 while isolate SAB E-41 had 98 % of homology level with B. amyloliquefaciens strain zy2 and isolate SAB E-57 had 97% of homology level with B. subtilis strain YRL02.

aureus.

PCR amplification of ketosynthase (KS) domain of PKS and adenylation (A) domain of NRPS were successfully amplified and sub-cloned into T-Vector pMD20. All isolates coded as SAB E-31, SAB E-41 and SAB E-57 possessed A domain and only two isolates coded as SAB E-41 and SAB E-57 possessed KS domain. DNA fragment encoding KS domain was in a size of 700 bp while for A domain was in a size of 1000

Sequences analysis of DNA fragment encoding KS domain using BlastX program indicated that isolate SAB E-41 showed a similarity level of 97% with type I PKS from Bacillus amyloliquefaciens LL3 and isolate SAB E-57 showed a similarity level of 98% with putative polyketide synthase pksL from B.

amyloliquefaciens subsp. plantarum CAU-B946 whereas for A domain indicated

that isolate SAB E-31 showed a similarity level of 81% with bacitracin synthetase 1from B. pumilus ATCC 7061. Isolate SAB E-41 s howed a similarity level of 80% with sur factin synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 and isolate SAB E-57 showed a similarity level of 81% with surfactin synthetaseA from the same strain of Bacillus, CAU-B946.

bp.

Keywords: Antimicrobial compound, fractionation, cloning, 16S rDNA, KS and A domain, Jaspis sp.