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
2012

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 university. Sources of information de rived or
quoted from published and unpublished works of other authors is mentioned in the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 gelcolumn 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 two
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. Fifteen of
the m named by BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA-7, BA-8, BA-11, BA12, 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 chloroformmethanol (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. Both of these active
compounds were successfully collected and showed antimicrobial activity against
S. aureus.
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.
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 bp.
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 CAUB946 and isolate SAB E-57 showed a similarity level of 81% with surfactin
synthetase A from the same strain of Bacillus, CAU-B946.

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

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written permission from Bogor Agricultural University

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
2012

External Examiner of the Thesis Examination Committee: Dr. Ir. Widanarni, M.Si

Title

Name
Student ID

: Antimicrobial Activity of Bioactive Compounds Isolated from
Marine Bacteria Associated with Sponge Jaspis sp. and Their
Genetics Analysis
: Effendi
: G351100021

Approved
Advisory Committee

Dr. Aris Tri Wahyudi, M.Si
Chairman

Dr. Munti Yuhana, M.Si
Committee Member

Agreed

Coordinator of Microbiology Mayor

Dr. Ir. Gayuh Rahayu

Date of Examination: August 15th , 2012

Dean of Graduate Schoo l

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

Date of Graduation:

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.Si 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 scientific 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.Si 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

Effendi

BIOGRAPHY
The author was born in Medan on August 21st , 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 author 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.
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
6th – 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 CONTENT................................................................................... 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
APPENDIXES ................................................................................................ 53

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
encoding A domain using BlastX Program ............................................... 28

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 F 254 eluted with n-butanol and ethyl acetate mixture (3:7).................. 19
6 Antimicrobial activity of active spots/fractions using
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 F 254 .................................................... 24
9 Antimicrobial activity of fifteen composite fractions purified
using PTLC Technique .............................................................................. 24
10 Gram staining o f three bacterial isolates coded as: A). SAB 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
A do main.................................................................................................... 31

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
from isolate SAB E-57 using BlastX program .......................................... 71

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). The increase of MDR cases has encouraged many
scientists to find the new bioactive compounds in order to solve the MDR problem.
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
chemotherape utic agents which can be developed and applied in pharmaceutical
industry in the future.
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).
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 microorganisms (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 microorganisms, 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 from the digestion of microbes as a food source
to mutualistic symbiosis with the spo nge. On the other hand, sponge is believed to
provide shelter from predators, a substrate for colonization, access to sunlight for
photosynthetic microbes 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 microorganisms 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 microorganisms in their tissues and the
growth of microorganisms 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 microorganisms 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 microorganisms 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 volution they have de velope d potent 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.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

Marine Sponges
Acanthela clethera
Aplisina sp.
Callyspongia sp.
Clathria bacilana
Clathria reinwardhi
Jaspis sp.
Phakelia aruensis
Phyllospongia sp.
Reniochalina sp.
Thionella cilindrica
Stylotella aurantiorum
Xestospongia sp.

Bacterial species
Flavobacterium sp., Aeromonas sp.
Aeromonas sp.
Pseudomonas sp.
Aeromonas sp.
Aeromonas sp.
Flavobacterum sp.
Bacillus sp., Aeromonas sp.
Vibrio sp., Pseudomonas sp., Aeromonas sp.
Acinetobacter sp.
Aeromonas sp.
Aeromonas sp., Vibrio 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 macroorganisms 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 vinylogous 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)
Pharmaceutical
Activity
Cytotoxic

Bioactive Compounds

Types of Sponge

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

important 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 Cmethyltransferase 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 domains 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 (5CAGGCCTAACACATGCAAGTC-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 compounds 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.
Extraction of Antimicrobial
Compounds

DNA Extraction

Antimicrobial Activity Test

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

Detection of Antimicrobial
Compounds

Fractionation of Bacterial
Crude Extract

Cloning of DNA Fragments
Encod ing KS and A Domain

Sequencing and Bioinformatics
Analysis of KS and A Domain

Purification of
Antimicrobial Compounds

Morphological and
Molecular Identification
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

hairdryer at 400 C. 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 1x10 6 CFU/ml, OD 620 0.45). The plate was incubated at 40 C 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 370 C. 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 F 254 ) 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 (450 C) containing test strains, and incubated at 37 0 C 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 F 254 ; 0.1 mm thickness) and eluted with nbutanol-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 chloroformmethanol-ethyl acetate solvent mixture (1:1:1). The active fractions that were
purified by preparative TLC were tested for their antimicrobial activity.

Morphological 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 94 0 C for 5 min, followed by 30 cycles of denaturation at 940 C for 1
min, annealing at 550 C for 1 min, elongation at 720 C 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 370 C for 30 min. After incubation, the
solution was added with 500 µl SDS 10% and 10 µl proteinase K and incubated
again at 370 C for 60 min. Afterwards, as much 80 µl NaCl was added together with
100 µl CTAB 10% and incubated at 650 C for 20 min. After incubation, added again
the solution with 650 µl PCI and shake n strongly 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 94 0 C for 5 min,
followed by 35 cycles of denaturation at 940 C for 1 min, annealing at 500 C for 1
min, elongation at 720 C for 1 min 10 sec and post PCR at 720 C 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 C for 1 min.
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 TaqTM (5 units/µl) and 28 µl milli Q. The

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 GENECLEAN ® II Kit.
Each of the purification prod ucts was sub-cloned into T-Vector pMD20 (

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