Journal of Coastal Development ISSN : 1410-5217

Volume 11 Number 3, June 2008 : 121-130

121
CORALS AS SOURCE OF BACTERIA WITH ANTIMICROBIAL
ACTIVITY

Ocky Karna Radjasa
1,2*
, Jutta Wiese
3
, Agus Sabdono
1,2
and Johannes F Imhoff
3

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

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

ABSTRACT

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

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

*Correspondence: Phone: +62-24-7460038; Fax: +62-24-7460039; e-mail : ocky _radjasa@yahoo.com




INTRODUCTION

Marine invertebrates that are mainly
accumulating within coral reef ecosystems
such as soft corals, sponges, tunicates, and
bryozoans have long been recognised as the
sources of structurally unique natural
products. However, it has been suggested
that natural products from marine
invertebrates have stricking similarities to
metabolites of their associated
microorganisms including bacteria (Proksch
et al. 2002; Imhoff and Sthr, 2003). Thus,
it is important to highlight the possible role
of bacteria in bacteria-invertebrate
associations in the production of
biologically active substances.
On the other hand, it has been well
known that corals harbor diverse microbial
communities (William et al. 1987; Shashar
et al. 1994; Kim, 1994; Santavy et al. 1995;
Kushmaro et al. 1996; Rohwer et al. 2001).
Every surface immersed in the marine
environment, including those organisms
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122
(Armstrong et al. 2001) such as muco-
polysaccharides-covered coral surfaces
providing a nutrient rich habitat for
heterotrophic bacteria that leading to the
formation of biofilm-forming microbial
communities (Kushmaro et al. 1997).
Inhibitory interactions among coral-
associated bacteria that occur on the coral
surface then could be of great interest to
search for secondary metabolite-producing
bacteria. In addition, examination concernin
secondary metabolite-producing bacteria
among coral colonizers have been strongly
neglected in comparison to soft-bodied
marine invertebrates.
Polyketides and non-ribosomal
peptides are two of the largest groups of
multifunctional proteins that create a
multitude of secondary metabolites
(Hutchinson, 2003), many of them are used
as therapeutic agents et al. 2001; Piel et al.
2003). Products of the microbial non-
ribosomal peptide synthesis include the
immunosuppressant cyclosporine and other
antibiotics such as gramicin S, tyrocin A and
surfactins (Kleinkauf and von Doehren,
1996). Among clinically important
polyketides are the antibiotibiotic
daunorubicin, erythromycin, lovastatin and
rapamycin (Due et al. 2001).
With advanced techniques of
molecular biology such as polymerase chain
reaction (PCR), it is now become possible to
carry out a screening on the presence of
polyketides and non ribosomal peptides by
using specific primers of polyketide
synthases (PKS) (Piel, 2002) and non
ribosomal polypeptide synthetases (NRPS)
(Marahiel et al. 1997).
The present study focusses on marine
bacteria, which are associated with the
corals representing different life forms
massive (Porites lutea), sub-massive
(Galaxea fascicularis), branching (Acropora
sp.) and folious (Pavona sp.) and produce
secondary metabolites with antimicrobial
activity. The examinations are coupled with
PCR-based analysis for the presence of
polyketide synthases and non-ribosomal
polypeptide synthetases of the bacterial
isolates.


MATERIALS AND METHODS

Sampling and isolation of coral-associated
bacteria

Four different corals were collected from the
vicinity of Panjang island, Jepara, North
Java Sea, Indonesia by scuba diving and
identified as Porites lutea, Galaxea
fascicularis, Acropora sp. and Pavona sp.
Upon collection coral fragments were put
into sterile plastic bags (Whirl-Pak, Nasco,
USA) and immediately brought to the
Marine Station of the Diponegoro University
where it was rinsed with sterile seawater and
scraped off with a sterile knife. The resultant
tissues were serially diluted in sterile
seawater, spread on Marine Broth, (Difco,
Becton Dickenson GmbH, Heidelberg,
Germany) and incubated at 20C for 48
hours. On the basis of morphological
features, colonies were randomly picked and
purified by making streak plates (Madigan et
al. 2000).

I nhibitory interaction tests

Inhibitory interaction tests of coral-
associated bacteria against test bacteria were
performed by using an overlay method. The
following bacteria were used: Escherichia
coli (DSM 498), Staphylococcus lentus
(DSM 6672), Bacillus subtilis (DSM 347),
and Candida glabrata (DSM 6425) obtained
from the German Culture Collection
(DSMZ, Braunschweig, Germany).
Overnight cultures of each target
microorganism in the logarithmic phase (ca.
10
9
cells ml
-1
) were mixed with TSB soft
agar medium (TSB 3g, NaCl 10g and Bacto-
agar 8g per liter). This mixture (1% v/v,
except C. glabrata of 10% v/v) was poured
on to the respective TSB medium surface,
which was previously inoculated with coral-
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123
associated bacteria and incubated for 4 d at
20C. The plates were then incubated at
20C for 48 hours. Antimicrobial activity
was defined by the formation of inhibition
zones around the bacterial colonies.

PCR-based analysis of NRPS and PKS
producing bacterial strains

To obtain genomic DNA of secondary
metabolite producing-strains for PCR
analysis, cell materials were taken from an
agar plate, suspended in sterile water
(Sigma, Germany) and subjected to five
cycles of freeze (-80C) and thaw (95C).
Amplification of peptide synthetase gene
fragments was carried out with the NRPS
degenerated primers A2gamF (5-AAG
GCN GGC GSB GCS TAY STG CC-3)
and A3gamR (5-TTG GGB IKB CCG GTS
GIN CCS GAG GTG-3) (Marahiel et al.,
1997) and PKS degenerated primers
KSDPQQF (5-MGN GAR GCN NWN
SMN ATG GAY CCN CAR CAN MG-3)
and KSHGTGR (5-GGR TCN CCN ARN
SWN GTN CCN GTN CCR TG -3) domain
(Piel, 2002). All primers were manufactured
by MWG-Biotech (Ebersberg, Germany).
PCR was performed with an
ProgeneThermal cycler (Techne,
Burkhardtsorf, Germany) as follows: 1 l
template DNA, 1 l of each of the
appropriate primers, and 23 l DNA free
water (Fluka, Sigma-Aldrich Chemie
GmbH, Germany) were added to puReTaq
Ready-To-Go PCR beads (Amersham
Biosciences Europe GmbH, Germany). The
NRPS-PCR run comprised 40 cycles with
denaturing conditions for 1 min at 95C,
annealing for 1 min at 70C and extension
for 2 min at 72C, respectively.
Pseudomonas sp. DSM 50117 was used as
positive control. The amplification of PKS
gene fragments included an initial
denaturating step at 94C for 2 min,
followed by 45 cycles at 94C for 1 min,
annealing at 55C for 1 min and elongation
at 72C for 2 min. Bacillus subtilis 168 as
utilized for positive control.
PCR amplification and sequencing of 16S
rRNA gene fragments.

PCR amplification of partial 16S rRNA gene
of active strains, purification of PCR
products and subsequent sequencing
analysis were performed according to the
method of Thiel and Imhoff (2003). The
almost complete 16S rDNA sequences of
strains were compared for homology to the
NCBI GenBank and EMBL databases using
BLAST (Basic Local Alignment Search
Tool) and FASTA searches (Altschul et al.,
1990, Pearson 1990). Sequences were
aligned using the program ClustalX Version
1.83 (Thomson et al. 1997). For
phylogenetic calculations the PhyML
software (Guindon and Gascuel, 2003) as
well as the online version of PhyML
(Guindon et al., 2005) were used. Trees
were calculated by Maximum Likelihood
(ML) method (Felsenstein, 1981) using the
GTR model and estimated proportion of
invariable sites as well as the Gamma
distribution parameter.

Cloning and sequencing of (putative)
peptide synthetase and polyketide
domains

The amplified PCR-products were purified
using the High Pure PCR Product
Purification Kit (Roche Diagnostics,
Mannheim, Germany) following the
manufacturers protocol. The Big Dye
Terminator Cycle Sequencing Ready
Reaction Kit (Applied Biosystems) was used
for subsequent sequencing on an ABI 310
analyzer (Perkin Elmer Applied Biosystems,
Foster City, USA).

DNA sequence accession numbers

The 16S rRNA gene sequences have been
entered into the GenBank database under the
sequence accession number AY000000-, the
putative peptide synthetase and polyketide
synthase sequences obtained under
AM287210-AM287213.
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124
RESULTS AND DISCUSSION

Results
I nhibitory interaction test

To estimate antimicrobial activity of coral
associated-bacteria, inhibitory interaction
tests between test strains and coral
associated isolates were carried out. As
shown in Table 1, a total of 13 bacterial
isolates belonging to the genera
Arthrobacter (1 strain), Bacillus (7 strains),
Micrococcus (1 strain), Pseudoalteromonas
(1 strain), Pseudovibrio (1 strain), and
Vibrio (2 strain), were successfully for their
inhibitorial effect against at least 1 test
strain. All 13 coral derived strains inhibited
the growth of the Gram-positive bacteria B.
subtilis. All isolates but one Pseudovibrio
isolate inhibited the second Gram-positive
test strain, S. lentus. Two Bacillus strains
showed an antibacterial activity against the
Gram-negative test strain E. coli, and one
Bacillus isolate showed an antifungal
activity against the yeast C. glabrata.

Detection of NRPS and PKS gene
fragments by PCR

PCR-based analysis using specific primers
of non-ribosomal peptide synthetase (NRPS)
and polyketide synthase (PKS) revealed that
the coral-associated bacterial strains Bacillus
sp BM1.5 and Micrococcus sp BJB showed
a PCR amplificate for NRPS. In addition,
two other bacterial strains (Bacillus subtilis
BJ.7 and Vibrio coralliitycus MJ.5)
exhibited a PCR product of PKS..

Putative peptide synthetase and
polyketide sequences.

Two PKS-amplifying bacterial strains (BJ.7
and MJ.5) had homologies with polyketide
synthase genes from Bacillus subtilis and
Anabaena sp 90 (Table 2), meanwhile the
other 2 NRPS-amplifying strains (BM1.5
and BJB) showed similarity with peptide
synthetase genes of Bacillus subtilis and
Actinoplanes teichomyceticus, respectively
(Table 3).

Discussion

In a study aimed at the search for
antimicrobial compounds from the sea, we
investigated bacteria associated with corals
from North Java Sea, Indonesia. Our
attention was focused on the examination of
coral associated-bacteria by using growth
inhibition tests followed by PCR-based
approach for the occurrence of gene
fragments of non-ribosomal peptide
synthetase (NRPS) and polyketide synthase
(PKS) which are believed to be responsible
for the biosynthesis of two main family of
secondary metabolites, polyketides and
peptides.
Microbes including bacteria play
crucial ecological roles in many marine
ecosystems such as tropical coral reefs.
Unfortunately, studies on bacterial
associations with corals have been much less
extensive (Knowlton and Rohwer, 2003).
Considering the intense interaction among
coral colonizers, in particular for the
competition for space and nutrient (Slattery
et al. 2001), coral associated-bacteria have
been considered as potential sources of
biologically active compounds (Moore,
1999).
In our study, we show that the corals
Porites lutea, Galaxea fascicularis,
Acropora sp. and Pavona sp. offered
promising potency as the source of coral
associates with biological activities against
E. coli, S. lentus, B. subtilis and C. glabrata.
A total of 13 bacterial isolates belonging to
Gram-positive genera, like Bacillus,
Micrococcus, and Arthrobacter, and to Gram
- negative genera, like Pseudoalteromonas,
Pseudovibrio and Vibrio were found to
inhibit the growth of at least 1 test strain.. It
is interesting to note that 7 out of 13 active
strains belonged to the genus Vibrio that
were isolated from corals Acropora sp (2),
G. fascicularis (3) and Pavona sp(2) but not
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125
from P. lutea. Furthermore, Acropora sp was
found to dominate the existence of active
strains including Bacillus (2), Micrococcus
(1), Arthrobacter (1) and Pseudovibrio (1).
One isolate obtained from the coral
Pavona sp. showed closest similarity to
Pseudoalteromonas sp. The genus of
Pseudoalteromonas is widely distributed in
the marine environment (Alexeeva et al.
2003) and considered one of the most
abundant group of marine -proteobacteria.
For the survival in such environments, the
members of genus Pseudoalteromonas have
been known to develop metabolic pathways
that produce biologically active metabolites.
Extensive studies regarding the capability of
Pseudoalteromonas to produce diverse
secondary metabolites were reported
(Austin, 1989; Jensen and Fenical, 1994;
Holmstrom and Kjelleberg, 1999). Further
work by (Reid et al. 1993; Deng et al.
1995) showed that Pseudoalteromonas
luteoviolacea, owns a non-ribosomal peptide
synthetase, which produces the siderophore
alterobactin. Radjasa et al. (2007) reported
the antibacterial activity of a bacterium
Pseudoaltermonas luteovioalacea TAB4.2
isolated from coral Acropora sp. collected
from the neighboring sampling site in the
North Java Sea. A novel antimicrobial
protein was also reported from a marine
bacterium strain X153 which was closely
related to P. piscicida (Longeon et al. 2004).
Furthermore, Sobolevskaya et al. (2005)
successfully isolated the recently decribed
Pseudoalteromonas maricaloris KMM 635
T
collected in the Coral Sea at a depth of 10 m
that produces a brominated cyclic
depsipeptides.
Hutchinson (2003) mentioned that
microorganisms, especially ones that inhabit
marine environments are noted for the
ability to produce a wide range of chemicals
known as secondary metabolites devoted to
the property of self-defense, intercellular
communication and other aspects of
microbial life. Du et al. (2001) mentioned
that polyketides and non-ribosomal peptides
represent the largest group of natural
products many of which are clinically
important drugs. For instance, non-
ribosomal peptides including cyclosporin,
penicillin and vancomycin are synthesized
by non-ribosomal peptide sythetases (NRPS)
(Kleinkauf and von Doehren, 1990). On the
contrary, polyketides such as daunorubicin,
erythromycin, lovastin and rapamycin are
derived from sequential condensation of
short carboxylic acids.
In the present study, however, only 2
strains amplified NRPS and 2 other strains
amplified PKS gene fragments Bacillus sp
BM1.5 showed closest similarity to the
genes for peptide synthetase and protein
building protein of Bacillus subtilis. Toh et
al. (2004) reported the occurrence of emetic
toxin of a foodborne pathogen, Bacillus
cereus that is putatively a product of non-
ribosomal peptide synthesis. The presence of
NRPS gene fragment in coral bacterium
Bacillus BM1.5 supported the fact that other
Bacillus specieroduce peptides using non-
ribosomal peptide synthesis (Stachelhaus
and Marahiel, 1995). Meanwhile another
bacterium associated with coral Acropora
sp, Bacillus subtilis BJ.7 was found to
amplify the gene fragment of PKS and
showed sequence similarity of 73% to
previously reported polyketide gene of
Bacillus subtilis (acc. no. U11039). Scotti et
al. (1993) studied the potential ability of B.
subtilis to synthesize polyketides.
Micrococcus sp BJB, had sequence
similarity to peptide synthetas of
Actinoplanes teichomyceticus.a The
members of Micrococcus are heterotrophic
bacteria distributed in various environments
such as seawater (Tanaka et al. 2001),
marine sediments (Zhong et al. 2002) and
prawn-rearing water (Phatarpekar et al.
2002). Members of the genus Micrococcus
have also the potency as producers of
antimicrobial compounds. A marine
bacterium Micrococcus MCCB 104 isolated
from hatchery water demonstrated
extracellular antagonistic properties against
Vibrio alginolyticus, V. parahaemolyticus, V.
fluviallis, V. nereis, V. proteolyticus, V.
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126
mediterranei, V. cholerae and Aeromonas sp
(Jayaprakash et al. 2005). Variacin, a
bacteriocin produced by Micrococcus
varians inhibited other Gram-posititve
bacteria, but not Gram-negative ones
(Pridmore et al, 1996).
Two antibacterial active isolates of
vibrios, V. coralliitycus MJ.5 and V.
parahaemolyticus MJ.11 were obtained
from coral Porites lutea. Isolate V.
coralliitycus MJ.5 also amplified the gene
fragment of PKS and had sequence
similarity (62%) to polyketide synthase of
Anabaena sp90. Polyketide natural products
are common metabolites of blue-green
algae (cyanobacteria) (Burdja et al. 2001).
Neilan et al. (1999) mentioned that
Cyanobacteria produced a myriad array of
secondary metabolites, including alkaloids,
polyketides, and non ribosomal peptides,
some of which are potent toxins. Isolate V.
parahaemolyticus MJ.11 inhibited the
growth of Bacillus subtilis and
Staphylococcus lentus. V. parahaemolyticus
has been reported as the member of Vibrio, a
dominant genus responsible for the observed
mortality in prawn-farmings (Lightner,
1988). As far as known it is uncertain, if V.
parahaemolyticus is also a pathogen of
corals. Cervino et al. (2004) considered that
further studies should include the
examination to determine if strains of V.
parahaemolyticus and V. alginolyticus are
capable of inducing disease signs in
Montastraea spp.


CONCLUSION

Our study on coral-associated bacteria
provides evidence of antimicrobial
properties among coral colonizers. Although
in minor numbers, the coral-associated
bacteria might be capable of producing
polyketides and peptides, because of the
detection of NRPS and PKS gene fragments
as well as their antimicrobial activities. The
discovery of the potential for the synthesis
of polyketides and peptides among coral-
associated bacteria should be of interest
because of promising applications in the
development of pharmacological
polyketides and peptides. Furthermore,
considering the fact that research on coral-
bacteria association has been fairly limited,
the future study on the search for secondary
metabolite producers among coral colonizers
should be given prominence.


ACKNOWLEDGEMENTS

This work was partly supported by the grant
from Ministry of Research and Technology,
Indonesia under the competitive research
grant scheme (RUT XI) No.
011.27.SK.RUT.2004. The work was also
part of a research grant provided by German
Center of Execellent within BIOTECmarin.
Ocky Karna Radjasa was research fellow of
the Alexander von Humboldt Foundation
(AvH) within the Georg Forster Research
Fellowship Programme.


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