Functional Gene-Guided Discovery of Type II Polyketides from Culturable Actinomycetes Associated with Soft Coral Scleronephthya sp.

Abstract

Compared with the actinomycetes in stone corals, the phylogenetic diversity of soft coral-associated culturable actinomycetes is essentially unexplored. Meanwhile, the knowledge of the natural products from coral-associated actinomycetes is very limited. In this study, thirty-two strains were isolated from the tissue of the soft coral Scleronephthya sp. in the East China Sea, which were grouped into eight genera by 16S rDNA phylogenetic analysis: Micromonospora, Gordonia, Mycobacterium, Nocardioides, Streptomyces, Cellulomonas, Dietzia and Rhodococcus. 6 Micromonospora strains and 4 Streptomyces strains were found to be with the potential for producing aromatic polyketides based on the analysis of KS a (ketoacyl-synthase) gene in the PKS II (type II polyketides synthase) gene cluster. Among the 6 Micromonospora strains, angucycline cyclase gene was amplified in 2 strains (A5-1 and A6-2), suggesting their potential in synthesizing angucyclines e.g. jadomycin. Under the guidance of functional gene prediction, one jadomycin B analogue (7b, 13-dihydro-7-O-methyl jadomycin B) was detected in the fermentation broth of Micromonospora sp. strain A5-1. This study highlights the phylogenetically diverse culturable actinomycetes associated with the tissue of soft coral Scleronephthya sp. and the potential of coral-derived actinomycetes especially Micromonospora in producing aromatic polyketides.

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Functional Gene-Guided Discovery of Type II Polyketides
from Culturable Actinomycetes Associated with Soft
Coral Scleronephthya sp
Wei Sun1, Chongsheng Peng2, Yunyu Zhao2, Zhiyong Li1*
1Marine Biotechnology Laboratory, State Key Laboratory of Microbial Metabolism & School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai,
People’s Republic of China, 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai, People’s Republic of China
Abstract
Compared with the actinomycetes in stone corals, the phylogenetic diversity of soft coral-associated culturable
actinomycetes is essentially unexplored. Meanwhile, the knowledge of the natural products from coral-associated
actinomycetes is very limited. In this study, thirty-two strains were isolated from the tissue of the soft coral Scleronephthya
sp. in the East China Sea, which were grouped into eight genera by 16S rDNA phylogenetic analysis: Micromonospora,
Gordonia, Mycobacterium, Nocardioides, Streptomyces, Cellulomonas, Dietzia and Rhodococcus. 6 Micromonospora strains and
4 Streptomyces strains were found to be with the potential for producing aromatic polyketides based on the analysis of KSa
(ketoacyl-synthase) gene in the PKS II (type II polyketides synthase) gene cluster. Among the 6 Micromonospora strains,
angucycline cyclase gene was amplified in 2 strains (A5-1 and A6-2), suggesting their potential in synthesizing angucyclines
e.g. jadomycin. Under the guidance of functional gene prediction, one jadomycin B analogue (7b, 13-dihydro-7-O-methyl
jadomycin B) was detected in the fermentation broth of Micromonospora sp. strain A5-1. This study highlights the
phylogenetically diverse culturable actinomycetes associated with the tissue of soft coral Scleronephthya sp. and the
potential of coral-derived actinomycetes especially Micromonospora in producing aromatic polyketides.
Citation: Sun W, Peng C, Zhao Y, Li Z (2012) Functional Gene-Guided Discovery of Type II Polyketides from Culturable Actinomycetes Associated with Soft Coral
Scleronephthya sp. PLoS ONE 7(8): e42847. doi:10.1371/journal.pone.0042847
Editor: Mark R. Liles, Auburn University, United States of America
Received June 5, 2012; Accepted July 12, 2012; Published August 7, 2012
Copyright: ? 2012 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (Grant No. 81102417) http://www.nsfc.gov.cn. The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: zyli@sjtu.edu.cn
Introduction
Corals are considered as the rainforests of the oceans. Coral-
derived natural products span a wide range of chemical classes (e.g.
prostaglandins, diterpenes, alkaloids and steroids) [1] and display a
variety of biological activities (e.g. antitumor, anti-inflammatory
and antibacterial activities) [2,3,4,5]. Actinomycetes are widely
distributed in marine habitats including the sea surface, water
column,marinesnow, sediments
[6,7,8,9,10,11,12]. Excitingly, many previously unknown actino-
mycete taxa have been successfully isolated from marine habitats
[7,13,14,15]. Meanwhile, novel and unique natural products have
beenincreasinglyrecovered
[16,17,18,19,20]. It has been demonstrated that some compounds
originally isolated from marine invertebrates are in fact produced
by microorganisms associated with invertebrates [21]. Actinomy-
cetes are frequent components of symbiotic communities in
invertebrates [6]. Since coral-associated actinomycetes could play
important role in protecting coral host [22], the actinomycetes
associated with corals may be involved in the synthesis of natural
products isolated from corals. Investigating the coral-associated
actinomycetes facilitates to reveal the true origin of biologically
active substances, and therefore, is significant for solving the
supply problem in marine drug development. However, to date,
related reports on coral-associated actinomycetes are still scarce
and mainly limited to stony corals [23,24,25]. Novel compounds
andmarineorganisms
from marine actinomycetes
with biological activity have been extracted from soft corals
[2,3,4,5], so, it is significant to investigate the soft coral-associated
actinomycetes regarding their diversity as well as their potential in
secondary metabolite biosynthesis.
Generally, traditional activity-based screening of microbial
strains and valuable natural products has its inherent limitation
because some natural products cannot be synthesized under the
normal condition or the compound yield is very low. With the
increasing knowledge of biosynthesis gene cluster for synthesizing
natural products, functional gene-based analysis provides a useful
approach for predicting natural products [26]. Gene-based
analysis has been previously applied in predicting type I polyketide
biosynthesis in marine Actinobacteria [27]. However, type II
polyketide biosynthesis has been rarely concerned. Aromatic
polyketides, which are synthesized by type II polyketide synthase
(PKS), exhibit a wide array of biological activities including
antibacterial, antitumor, antiviral and enzyme inhibitory activities
[28], and afford some of the most common antibiotics and anti-
cancer drugs currently in clinical use, e.g. tetracyclines and
anthracyclines. Type II PKS consists of three or more enzymes
that act in an iterative manner. The core module in all type II PKS
gene clusters is composed of ketoacyl-synthase (KSa), chain length
factor (KSb) and acyl carrier protein (ACP). Moreover, cyclase is
responsible for the cyclization of aromatic polyketides. Thus, KSa
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and cyclase gene can be used as makers for the screening of type II
polyketide compounds.
With the aim to reveal the diversity of culturable Actinobacteria
associated with soft coral and screen the actinomycetes with the
potential for synthesizing type II polyketides, actinomycetes were
isolated from the tissue of soft coral Scleronephthya sp. in the East
China Sea. The isolates were tested for their potential in
producing aromatic polyketides by the detection of KSa and
cyclase gene. Finally, type II polyketide compound was identified
in the fermentation broth of Micromonospora sp. strain A5-1 under
the guidance of functional gene prediction.
Methods
Ethics Statement: N/A
This study was approved by Shanghai Jiao Tong University,
China.
Sample collection and isolation of actinomycetes
Soft coral Scleronephthya sp. was collected from Zhao’an Bay
(23u539N; 117u109E) in the East China Sea. The sample was
stored at 220uC until analysis. Coral tissue was rinsed three times
with sterile artificial seawater (ASW) (1.1 g CaCl2, 10.2 g
MgCl2?6H2O, 31.6 g NaCl, 0.75 g KCl, 1.0 g Na2SO4, 2.4 g
Tris-HCl, 0.02 g NaHCO3, 1L distilled water, pH 7.6) to remove
the microbes loosely attached on the surface, and then aseptically
grinded using a pestle and a mortar. Six types of media were used
for isolating coral-associated actinomycetes [7,10,12,29] (Table
S1). All media were supplemented with K2Cr2O7(50 mg ml21) to
inhibit the growth of fungi, and with nalidixic acid (15 mg ml21) to
inhibit fast-growing Gram-negative bacteria. Actinomycetes were
isolated by serial dilution on agar plates in triplicate at 28uC for 3–
6 weeks. The colonies bearing distinct morphological character-
istics were picked up and transferred onto freshly prepared plates
until pure cultures were obtained.
Genomic DNA extraction
A single colony was transferred to a 5-ml microtube with 1 ml of
liquid medium from which the isolate was originally picked up.
The cultures were incubated for 3–5 days at 28uC with shaking at
180 rpm. Microbial cells were collected by centrifugation and
genomic DNA was extracted as described by Li and De Boer [30].
PCR amplification of 16S rRNA gene
Theuniversal
GATCCTGGCTCAG-39) and 1500R (59-AGAAAGGAGGT-
GATCCAGCC-39) were used for the amplification of 16S rRNA
gene [31]. The PCR was carried out in a 20 ml PCR mixtures
including 10 ml Taq Premix (Takara, Dalian, China), 0.5 ml 27F
(10 mM), 0.5 ml 1500R (10 mM) and 5% DMSO. Cycling
conditions were as follows: initial denaturation at 95uC for
3 min, 30 cycles of 94uC for 30 s, 54uC for 40 s, and 72uC for
2 min, and a final extension of 10 min at 72uC.
bacterial primers27F(59-GAGTTT-
PCR amplification of KSaand angucycline cyclase gene
The degenerate primers IIPF6 (59-TSGCSTGCTTCGAYGC-
SATC-39) and IIPR6 (59-TGGAANCCGCCGAABCCGCT-39)
were employed to amplify the KSagene of PKS II [32]. The PCR
was performed in a 20 ml PCR mixtures including 10 ml Taq
Premix, 0.8 ml IIPF6 (25 mM), 0.8 ml IIPR6 (25 mM) and 5%
DMSO. Cycling conditions were as follows: initial denaturation at
95uC for 5 min, 30 cycles of 95uC for 35 s, 55uC for 40 s, and
72uC for 1 min, and a final extension of 10 min at 72uC. The
degenerate primers AuF3 (59-GAACTGGCCSCGSRTBTT-39)
and AuR4 (59-CCNGTGTGSARSKTCATSA-39) were applied
in the amplification of angucycline cyclase gene [33]. 20 ml PCR
mixtures included 10 ml Taq Premix, 1 ml AuF3 (40 mM), 1 ml
AuR4 (40 mM) and 5% DMSO. Cycling conditions were as
follows: initial denaturation at 94uC for 5 min, 30 cycles of 94uC
for 45 s, 60uC for 1 min, and 72uC for 1 min, and a final
extension of 8 min at 72uC.
Sequencing and phylogenetic analyses
The PCR products were purified using Agarose Gel DNA
Purification Kit (Takara, Dalian, China) and sequenced on an ABI
3730 automated sequencer by Beijing Genomic Institute (Shenz-
hen, China). The gene sequences obtained were proofread using
Chromas, version 1.62 (Technelysium). The nucleotide sequences
were matched with published sequences in NCBI using the
BLAST search program (http://www.ncbi.nlm.nih.gov/). For
KSaand cyclase gene, translated protein sequences were derived
from nucleotide sequences using the ORF FINDER available at
the NCBI (http://www.ncbi.nlm.nih.gov/projects/gorf/). The
deduced amino acid sequences were used as queries to search
the related proteins in the nr protein database using the BLASTP
algorithm. For 16S rRNA gene and KSa, multiple sequence
alignment was performed using CLUSTALX. Phylogenetic tree
was constructed using Mega 4 [34]. The consistency of the trees
was verified by bootstrapping (1,000 replicates) for parsimony.
Nucleotide sequence accession numbers
16S rRNA, KSa (PKS II) and angucycline cyclase gene
sequences from the soft coral-derived actinomycete isolates were
deposited in the GenBank database under the following accession
numbers: JN627163–JN627194,
JQ943912–JQ943913.
JN627195–JN627204 and
Fermentation and chemical identification
Strain A5-1 was inoculated in 25 ml flask using GYMM
medium (20 g glycerol, 10 g yeast extract, 4 g malt extract, 10 g
mannitol, 1 liter ASW) at 28uC,180 rpm in the dark for 3 weeks,
and then transferred to a 250 ml Erlenmeyer flask containing
100 ml of D-galactose-L-isoleucine medium [35]. The culture was
incubated at 28uC, 180 rpm in the dark for 45 days. On the
fifteenth day, ethanol was added to a final concentration of 6% (v/
v) to induce the synthesis of jadomycin [35].
After mycelium was removed by filtration, the fermentation
broth was extracted with 100 ml of acetic ether (EtOAc) and
concentrated in vacuo. EtOAc extract was dissolved in methanol
for HPLC-DAD analysis on an Agilent 1200 (Agilent Technolo-
gies, USA) series with an on-line Diode Array Detector (DAD/
UV)andaC18
RP-column
4.66150 mm). Ultraviolet absorption was compared with that of
jadomycins according to their maximum absorption wavelength
(lmax) [36].
For LC-QTOF-MS analysis, the methanol solution of strain A5-
1 extract was detected on an ultra performance liquid &
quadrupole time of flight mass spectroscopy (UPLC-QTOF-MS
Premier, Waters Corporation, USA). The analytes were separated
onaC18
RP-column(ACQUITY
2.16100 mm, Waters Co.), with linear gradient elution from
H2O (1% formic acid) to 35% H2O/MeCN (1% formic acid).
Total ions chromatography (TIC) and mass spectrum of selected
ion were acquired in positive electro-spray ionization mass
spectrum (ESI-MS) mode.
In the case of1H NMR analysis, the EtOAc extract was dried in
vacuo and then dissolved in CD3OD. Proton nuclear magnetic
(EclipseXDB-C18
5 mm,
BEH-C18
1.7 mm,
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resonance (1H NMR) spectrum was recorded on an AVANCE III
400 spectrometer (400 MHz, Bruker).
Results
Recovery and phylogenetic diversity of coral
Scleronephthya sp.-associated actinomycetes
After incubation for 6 weeks, 32 isolates were recovered. Based
on the BLAST analyses of 16S rRNA gene sequences, these 32
isolates were assigned to Actinobacteria with 98–100% similarity to
their nearest relatives in the GenBank database, including 8
genera: Micromonospora (8 isolates), Gordonia (8 isolates), Mycobacte-
rium (6 isolates), Nocardioides (3 isolates), Streptomyces (4 isolates),
Cellulomonas (1 isolate), Dietzia (1 isolate) and Rhodococcus (1 isolate)
(Table 1; Figure 1), which indicated that Micromonospora and
Gordonia are relatively dominant among the culturable actinomy-
cetel community in the tissue of the soft coral Scleronephthya sp..
Four strains (Gordonia sp. strain A5-14, Rhodococcus sp. strain A2-19,
Micromonospora sp. strains A1-11 and A5-2) share high homology
with relatives derived from marine sediments. Eight strains
(Mycobacterium poriferae strains A1-12, A1-17, A3-1, A3-11 and
A5-20, Micromonospora sp. strains A5-1, A6-2 and A6-10) show high
similarity to relatives isolated from marine sponges.
Notably, significant differences in the total number of isolates
were observed among the 6 different media (Figure S1). M5
produced the highest recovery with 10 isolates, followed by M1 (8
Figure 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequence (ca.1,400 bp) of actinomycetes from the tissue of
soft coral Scleronephthya sp. The sequences obtained in this work are marked by black dot. The number is the percentage indicating the level of
boot strap support, based on a neighbor-joining analysis of 1,000 resampled data sets. The scale bar represents 0.02 substitutions per nucleotide
position.
doi:10.1371/journal.pone.0042847.g001
Table 1. Actinomycetes and those with PKS II gene from the soft coral Scleronephthya sp.
GenusStrain (NCBI accession no.) Most closely related strain (NCBI accession no.)Identity (%) PKS II
CellulomonasA5-19 (JN627163) Cellulomonas sp.(EU303275)98
2
Dietzia A1-8 (JN627164)D. maris (GQ870425)99
2
Gordonia A1-3 (JN627165) G. paraffinivorans (NR_028832) 99
2
A1-10 (JN627166)G. paraffinivorans (NR_028832) 99
2
A4-4 (JN627167)G. paraffinivorans (NR_028832)99
2
A4-16 (JN627168)G. paraffinivorans (NR_028832) 99
2
A4-20 (JN627169) G. lacunae (GU727686) 99
2
A5-8 (JN627170)G. bronchialis (FJ536306) 99
2
A5-9 (JN627171)G. alkanivorans (AY995556)99
2
A5-14 (JN627172)Gordonia sp. (DQ448772) 99
2
MycobacteriumA1-12 (JN627173)M. poriferae (NR_025235) 99
2
A1-17 (JN627174) M. poriferae (NR_025235) 99
2
A3-1 (JN627175)M. poriferae (NR_025235) 99
2
A3-11 (JN627176) M. poriferae (NR_025235)99
2
A5-20 (JN627177)M. poriferae (NR_025235) 99
2
A5-7 (JN627178) M. duvalii (NR_026073)100
2
RhodococcusA2-19 (JN627179)Rhodococcus sp. (GQ871747)99
2
NocardioidesA1-2 (JN627180)Nocardioides sp. (FJ711223)99
2
A5-6 (JN627181)Nocardioides sp. (FJ711223)98
2
A6-8 (JN627182)Nocardioides sp. (FJ711223)99
2
StreptomycesA4-1 (JN627183)S. variabilis (EU841659)99
+
+
+
+
A4-2 (JN627184)S. variabilis (EU841659)100
A4-3 (JN627185) S. variabilis (EU841659) 99
A6-1 (JN627186)
S. variabilis (EU841659)99
MicromonosporaA1-11 (JN627187) Micromonospora sp. (EU214967) 99
2
A1-15 (JN627188)Micromonospora sp. (EU531460)99
+
+
+
+
+
+
A5-2 (JN627189)Micromonospora sp. (EU214980) 99
A5-13 (JN627190)M. purpureochromogenes (FJ486489)100
A5-1 (JN627191)Micromonospora sp. (GU002071)99
A6-2 (JN627192)Micromonospora sp. (GU002071)99
A6-9 (JN627193)Micromonospora sp. (EU531460)100
A6-10 (JN627194) Micromonospora sp. (GQ863921)99
2
doi:10.1371/journal.pone.0042847.t001
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isolates), M4 (6 isolates), M6 (5 isolates), M3 (2 isolates) and M2 (1
isolate). Additionally, the actinomycete diversity recovered from
the different media varied (Figure S1). For example, M1 and M5
yielded the highest diversity with 5 genera, followed by M6 (3
genera), M4 (2 genera), M2 (1 genus) and M3 (1 genus). As
expected, the combination of 6 media achieved a better
recoverability of coral-associated actinomycetes.
The potential for producing type II polyketides based on
functional gene analysis
The presence of KSagene was detected in two of the eight
genera, Streptomyces (4 strains) and Micromonospora (6 strains)
(Table 1). Based on BLAST analyses, the KSasequences from
four Streptomyces strains show high (98.4–98.8%) sequence similarity
to their BLAST matches, whereas, the KSasequences from six
Micromonospora strains share relatively lower (,89.4%) homology
with previously reported sequences.
A phylogenetic tree was generated using 10 KSaamino acid
sequences obtained in this study and 17 reference sequences
retrieved from GenBank (Figure 2). Reference sequences related to
biosynthetic pathways help to group the obtained sequences into
different clusters representing different chemotypes. As shown in
Fig. 2, KSasequences from 6 Micromonospora strains are separated
into three major phylogenetic divisions. For example, sequences
from strains A5-1 and A6-2 fall into a cluster with angucycline
ketosynthase sequences, and show the closest evolutionary
relationship with Jad A (AAB36562) which is involved in the
biosynthesis of jadomycin B (Table 2). Sequences of strains A5-2,
A6-9 and A5-13 are clustered in a group together with relative Lac
31 (ABX71114) associated with the biosynthetic pathway of
lactonamycin. Interestingly, the unique KSasequence from strain
A1-15 is clearly separated from any known sequence involved in
characterized pathways. After the phylogenetic analysis, 32 strains
were tested for the presence of angucycline cyclase gene which is
involved in the aromatization of angucycline. The target band of
Figure 2. Neighbor-joining tree constructed using aligned KSadomain amino acid sequence (203 amino acid positions) from type II
PKSs. The sequences obtained in this work are marked by black dot. Next to the taxon name, GenBank accession number of KSadomain amino acid
sequence or/and the identified compounds are indicated. Bootstrap values calculated from 1,000 resamplings using neighborjoining are shown at
the respective nodes when the calculated values were 50% or greater. The scale bar represents 0.05 substitutions per amino acid position.
doi:10.1371/journal.pone.0042847.g002
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approximately 650 bp was successfully amplified in Micromonospora
sp. strains A5-1 and A6-2. This result indicates that these two
Micromonospora strains have the potential in producing angucycline
compounds such as jadomycin.
The identification of a novel analogue of jadomycin B in
the fermentation broth of Micromonospora sp. strain A5-1
Among Micromonospora sp. strains A5-1 and A6-2 with potential
to produce jadomycin B or its analogues, strain A5-1 was selected
Table 2. KSanucleotide sequences.
StrainNCBI accession no.Top BLAST match (NCBI accession no.)Identity (%)
Micromonospora sp. A1-15JN627200 Streptomyces sp. SirexAA-E b-ketoacyl synthase gene
(CP002993)
85.8
Micromonospora sp. A5-1 JN627196Micromonospora sp. SAUK6030 type II polyketide
synthase-like gene (GQ118939)
86.9
Streptomyces venezuelae jadomycin polyketide
ketosynthase (jadA) gene (AF126429)
85.8
Micromonospora sp. A5-2 JN627201Micromonospora aurantiaca ATCC 27029 b-ketoacyl
synthase gene (CP002162)
88.2
Micromonospora sp. A5-13 JN627202Micromonospora aurantiaca ATCC 27029 b-ketoacyl
synthase gene (CP002162)
88.9
Micromonospora sp. A6-2JN627203Micromonospora sp. SAUK6030 type II polyketide
synthase-like gene (GQ118939)
86.7
Streptomyces venezuelae jadomycin polyketide
ketosynthase (jadA) gene (AF126429)
85.8
Micromonospora sp. A6-9JN627204Micromonospora aurantiaca ATCC 27029 b-ketoacyl
synthase gene (CP002162)
89.4
Streptomyces sp. A4-1JN627199Streptomyces sp. JS-14 ketosynthase gene (GU373728)98.7
Streptomyces sp. A4-2JN627198Streptomyces sp. JS-14 ketosynthase gene (GU373728)98.8
Streptomyces sp. A4-3 JN627197Streptomyces sp. JS-14 ketosynthase gene (GU373728)98.5
Streptomyces sp. A6-1JN627195 Streptomyces sp. JS-14 ketosynthase gene (GU373728)98.4
doi:10.1371/journal.pone.0042847.t002
Figure 3. HPLC of the EtOAc extract of Micromonospora sp. strain A5-1 fermentation broth (UV spectra of selected peaks at tR
5.22 min show similar absorption as jadomycins).
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Figure 4. TIC of the EtOAc extract of Micromonospora sp. strain A5-1 fermentation broth (the peak with tRat 4.18 min is putative
jadomycin analogue).
doi:10.1371/journal.pone.0042847.g004
Figure 5. Mass spectrum of selected ion at tR4.18 min in TIC.
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for fermentation to test the gene prediction since the two strains
belong to the same species. Only 10 mg EtOAc extract of the
fermentation broth of Micromonospora sp. strain A5-1 was obtained
because Micromonospora sp. strain A5-1 grew very slowly and the
biomass was very low.
Jadomycin B displays 5 UV absorptions: 212 nm, 238 nm,
280 nm, 312 nm and 520 nm [36]. In the EtOAc extract of
fermentation broth of Micromonospora sp. strain A5-1, one peak
(retention time (tR) at 5.22 min, Figure 3) shows similar UV
profiles as that of jadomycins except the absorption band over
350 nm which is contributed by the substructure of p-quinone.
The result suggests the existence of jadomycin B analogue with
one keto function reduction in the fermentation broth of
Micromonospora sp. strain A5-1.
Jadomycin B shows mass to charge (m/z) at 306 and 550 in ESI
mass spectrum which are assigned as key fragmentation ion
[phenanthroviridin+H]+and pseudomolecular ion [jadomycin
B+H]+[37]. In this study, TIC of the EtOAc extract of
Micromonospora sp. strain A5-1 fermentation broth shows one m/z
566 with tRat 4.18 min (Figure 4), which is 16 amu more than
that of pseudo-molecular ion of jadomycin B. So, the 14 amu
corresponding to methylene should be added to the keto reduction
derivative of jadomycin B. In the mass spectrum (Figure 5), the key
fragmentation ion at m/z 322 instead of that at m/z 306 of
jadomycins supports the change in phenanthroviridin. Based on
the spectral data analysis and comparison with jadomycin B, the
putative structure of target compound corresponding to the peak
with tRat 4.18 min in Figure 4 should be 7b, 13-dihydro-7-O-
methyl jadomycin B. The possible MS fragmentations are shown
in Figures 5 & 6. Meanwhile, this assignment is also supported by
the1H NMR data (Figure 7), which are consistent with that of
jadomycin B [36].
Figure 6. Suggested fragmentation process of selected ion at tR4.18 min in TIC.
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Discussion
The phylogenetic diversity of culturable actinomycetes
associated with coral Scleronephthya sp.
Studies on sponge-associated actinomycetes indicate that
medium exhibits significant effect on the diversity of Actinobacteria
recovered [12,38]. So, in order to gain a better recoverability of
coral-associated actinomycetes, six different media were used in
this study. Similarly, medium-dependent recovery efficiency was
observed. Taking the dominant Micromonospora for example, it was
recovered from only 3 types of media. Moreover, not any one
medium can recover all 8 genera, suggesting the necessity of
combining different media to increase the recovery rate of cultured
actinomycetes.
Prior to this study, the investigation of culturable actinomycetes
has been mainly focused on stony corals [23,24,25]. In this study, a
total of 8 genera were successfully isolated from the soft coral
Scleronephthya sp., including Micromonospora, Gordonia, Mycobacterium,
Nocardioides, Streptomyces, Cellulomonas, Dietzia and Rhodococcus. The
culturable actinomycetes include both common and rare actino-
mycetes species. Rare actinomycetes derived from marine habitats,
such as Salinispora [39], Verrucosispora [40] and Micromonospora
[41,42,43], have shown their unique capacity to produce novel
natural products. BLAST analyses shows that the isolated
actinomycete strains e.g. Micromonospora, Mycobacterium, Gordonia
and Rhodococcus have closest relatives derived from marine sponges
or marine sediments. Mycobacterium poriferae was originally isolated
from the sponge Halichondria bowerbanki [44]. Recently, 11 strains of
M. poriferae have been isolated from the sponge Amphimedon
queenslandica and the authors proposed that the isolates may
represent a sponge-specific phylotype [45]. It is worth noting that,
in this study, 5 strains M. poriferae were isolated from the tissue of
this soft coral, suggesting that M. poriferae are not merely limited in
sponges.
The potential of culturable actinomycetes associated
with coral Scleronephthya sp. in producing type II
polyketides
It is proposed that actinomycetes with PKS gene do produce a
larger number of new metabolites [26]. In this study, actinomy-
cetes with the potential to produce aromatic polyketides were
screened by detecting KSaand cyclase genes of PKS II. Among
the 32 strains actinomycetes, 10 strains from two genera
Streptomyces and Micromonospora yielded positive results. Streptomyces
is a well-known polyketide producer, so it is not surprising that
KSagene was identified in all the 4 Streptomyces strains. Prior to this
study, it was found that most of the Micromonospora strains are not
potential producers of type II polyketides [26,33]. The known
secondary metabolites produced by Micromonospora are mainly
aminoglycosides, macrolides and enediynes, few aromatic polyke-
tides are known to be produced by Micromonospora except
Figure 7.1H NMR data of selected ion at tR4.18 min in TIC.
doi:10.1371/journal.pone.0042847.g007
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Page 10

anthracyclines [46]. In contrast, it is unexpected that the target
gene was detected in 6 of 8 Micromonospora strains, indicating that
some coral-associated Micromonospora strains have the potential in
producing aromatic polyketides.
Early in 1994, it was known that the production of jadomycin B
in Streptomyces venezuelae ISP5230 needed to be induced by heat
shock, ethanol treatment or phage infection [35]. Apparently, the
jadomycin pathway is cryptic and only activated under specific
conditions. In this case, natural product discovery strategy based
on traditional bioassay is limited. Similarly, the D-galactose-L-
isoleucine medium, which is beneficial for producing jadomycin B
[35], was used in the fermentation of Micromonospora sp. strain A5-
1, followed with ethanol induction [35]. Although jadomycin B
was not found in the fermentation broth of Micromonospora sp.
strain A5-1, a novel analogue of jadomycin B, i.e. 7b, 13-dihydro-
7-O-methyl jadomycin B, was identified, which proved the
prediction based on the functional gene screening. This study
indicates that gene-based screening may guide the discovery of
target metabolites especially those cannot be synthesized under the
normal cultivation conditions. However, because Micromonospora
sp. strain A5-1 grew very slowly and the yield of target compound
was very low, so, in this study, the pure 7b, 13-dihydro-7-O-
methyl jadomycin B was not isolated successfully. Alternatively, for
the slowly-growing Micromonospora with type II polyketides
producing potential, the cloning and heterologous expression of
related gene cluster is a potential choice for future investigation.
The results from this study indicate that the soft coral tissue
harbors diverse actinomycetes, some of which are with potential in
synthesizing type II polyketides. This study, together with
actinomycetes from stony corals [23,24,25,47], suggests that the
diverse culturable coral-associated actinomycetes are important
source for marine natural products.
Supporting Information
Table S1
from the soft coral Scleronephthya sp.
(DOC)
Media used for the isolation of actinomycetes
Figure S1
using six media.
(DOC)
The diversity of actinomycetes recovered
Author Contributions
Conceived and designed the experiments: ZL WS. Performed the
experiments: WS YZ. Analyzed the data: WS ZL CP. Wrote the paper:
WS ZL CP.
References
1. Blunt JW, Copp BR, Hu WP, Munro MHG, Northcote PT, et al. (2009) Marine
natural products. Nat Prod Rep 26: 170–244.
2. Yan XH, Gavagnin M, Cimino G, Guo YW (2007) Two new biscembranes with
unprecedented carbon skeleton and their probable biogenetic precursor from the
Hainan soft coral Sarcophyton latum. Tetrahedron Lett 48: 5313–5316.
3. Han L, Wang CY, Huang H, Shao CL, Liu QA, et al. (2010) A new pregnane
analogue from Hainan soft coral Scleronephthya gracillimum Ku ¨kenthal. Biochem
Syst Ecol 38: 243–246.
4. Li L, Sheng L, Wang CY, Zhou YB, Huang H, et al. (2011) Diterpenes from the
Hainan soft coral Lobophytum cristatum Tixier-Durivault. J Nat Prod 74: 2089–
2094.
5. Yan XH, Liu HL, Huang H, Li XB, Guo YW (2011) Steroids with aromatic A-
rings from the Hainan soft coral Dendronephthya studeri Ridley. J Nat Prod 74:
175–180.
6. Ward AC, Bora N (2006) Diversity and biogeography of marine actinobacteria.
Curr Opin Microbiol 9: 279–286.
7. Mincer TJ, Jensen PR, Kauffman CA, Fenical W (2002) Widespread and
persistent populations of a major new marine actinomycete taxon in ocean
sediments. Appl Environ Microb 68: 5005–5011.
8. Bredholdt H, Galatenko OA, Engelhardt K, Fjaervik E, Terekhova LP, et al.
(2007) Rare actinomycete bacteria from the shallow water sediments of the
Trondheim fjord, Norway: isolation, diversity and biological activity. Environ
Microbiol 9: 2756–2764.
9. Anzai K, Nakashima T, Kuwahara N, Suzuki R, Ohfuku Y, et al. (2008)
Actinomycete bacteria isolated from the sediments at coastal and offshore area of
nagasaki prefecture, Japan: diversity and biological activity. J Biosci Bioeng 106:
215–217.
10. Zhang HT, Lee YK, Zhang W, Lee HK (2006) Culturable actinobacteria from
the marine sponge Hymeniacidon perleve: isolation and phylogenetic diversity by
16S rRNA gene-RFLP analysis. Antonie van Leeuwenhoek 90: 159–169.
11. Jiang SM, Sun W, Chen MJ, Dai SK, Zhang L, et al. (2007) Diversity of
culturable actinobacteria isolated from marine sponge Haliclona sp.. Antonie van
Leeuwenhoek 92: 405–416.
12. Abdelmohsen UR, Pimentel-Elardo SM, Hanora A, Radwan M, Abou-El-Ela
SH, et al. (2010) Isolation, phylogenetic analysis and anti-infective activity
screening of marine sponge-associated actinomycetes. Mar Drugs 8: 399–412.
13. Zhang HT, Zheng W, Huang JY, Luo HL, Jin Y, et al. (2006) Actinoalloteichus
hymeniacidonis sp. nov., an actinomycete isolated from the marine sponge
Hymeniacidon perleve. Int J Syst Evol Micr 56: 2309–2312.
14. Olson JB, Harmody DK, Bej AK, McCarthy PJ (2007) Tsukamurella spongiae sp.
nov., a novel actinomycete isolated from a deep-water marine sponge. Int J Syst
Evol Micr 57: 1478–1481.
15. Xiao J, Luo YX, Xie SJ, Xu J (2011) Serinicoccus profundi sp. nov., an actinomycete
isolated from deep-sea sediment, and emended description of the genus
Serinicoccus. Int J Syst Evol Micr 61: 16–19.
16. Bull AT, Stach JEM (2007) Marine actinobacteria: new opportunities for natural
product search and discovery. Trends Microbiol 15: 491–499.
17. Macherla VR, Liu J, Sunga M, White DJ, Grodberg J, et al. (2007)
Lipoxazolidinones A, B, and C: antibacterial 4-oxazolidinones from a marine
actinomycete isolated from a Guam marine sediment. J Nat Prod 70: 1454–
1457.
18. Martin GDA, Tan LT, Jensen PR, Dimayuga RE, Fairchild CR, et al. (2007)
Marmycins A and B, cytotoxic pentacyclic C-glycosides from a marine sediment-
derived actinomycete related to the genus Streptomyces. J Nat Prod 70: 1406–
1409.
19. Khan ST, Komaki H, Motohashi K, Kozone I, Mukai A, et al. (2011) Streptomyces
associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary
metabolites and products. Environ Microbiol 13: 391–403.
20. Li K, Li QL, Ji NY, Liu B, Zhang W, et al. (2011) Deoxyuridines from the
marine sponge associated actinomycete Streptomyces microflavus. Mar Drugs 9:
690–695.
21. Radjasa OK, Vaske YM, Navarro G, Vervoort HC, Tenney K, et al. (2011)
Highlights of marine invertebrate-derived biosynthetic products: Their biomed-
ical potential and possible production by microbial associants. Bioorg Med
Chem 19: 6658–6674.
22. Nithyanand P, Thenmozhi R, Rathna J, Pandian SK (2010) Inhibition of
Streptococcus pyogenes biofilm formation by coral-associated actinomycetes. Curr
Microbiol 60: 454–460.
23. Lampert Y, Kelman D, Dubinsky Z, Nitzan Y, Hill RT (2006) Diversity of
culturable bacteria in the mucus of the Red Sea coral Fungia scutaria. FEMS
Microbiol Ecol 58: 99–108.
24. Nithyanand P, Pandian SK (2009) Phylogenetic characterization of culturable
bacterial diversity associated with the mucus and tissue of the coral Acropora
digitifera from the Gulf of Mannar. FEMS Microbiol Ecol 69: 384–394.
25. Nithyanand P, Manju S, Pandian SK (2011) Phylogenetic characterization of
culturable actinomycetes associated with the mucus of the coral Acropora digitifera
from Gulf of Mannar. FEMS Microbiol Lett 314: 112–118.
26. Schneemann I, Nagel K, Kajahn I, Labes A, Wiese J, et al. (2010)
Comprehensive investigation of marine actinobacteria associated with the
sponge Halichondria panicea. Appl Environ Microb 76: 3702–3714.
27. Gontang EA, Gaude ˆncio SP, Fenical W, Jensen PR (2010) Sequence-based
analysis of secondary-metabolite biosynthesis in marine actinobacteria. Appl
Environ Microb 76: 2487–2499.
28. Hertweck C, Luzhetskyy A, Rebets Y, Bechthold A (2007) Type II polyketide
synthases: gaining a deeper insight into enzymatic teamwork. Nat Prod Rep 24:
162–190.
29. Webster NS, Wilson KJ, Blackall LL, Hill RT (2001) Phylogenetic diversity of
bacteria associated with the marine sponge Rhopaloeides odorabile. Appl Environ
Microb 67: 434–444.
30. Li X, De Boer SH (1995) Selection of polymerase chain reaction primers from
an RNA intergenic spacer region for specific detection of Clavibacter michiganensis
subsp. sepedonicus. Phytopathol 85: 837–842.
31. Woese CR, Gutell R, Gupta R, Noller HF (1983) Detailed analysis of the higher-
order structure of 16S-like ribosomal ribonucleic acids. Microbiol Rev 47: 621–
669.
32. Metsa ¨-Ketela ¨ M, Salo V, Halo L, Hautala A, Hakala J, et al. (1999) An efficient
approach for screening minimal PKS genes from Streptomyces. FEMS Microbiol
Lett 180: 1–6.
Coral Actinomycetes
PLoS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e42847

Page 11

33. Ouyang YC, Wu HB, Xie LW, Wang GH, Dai SK, et al. (2011) A method to
type the potential angucycline producers in actinomycetes isolated from marine
sponges. Antonie van Leeuwenhoek 99: 807–815.
34. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular evolutionary
genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599.
35. Doull JL, Singh AK, Hoare M, Ayer SW (1994) Conditions for the production of
jadomycin B by Streptomyces venezuelae ISP5230: effects of heat shock, ethanol
treatment and phage infection. J Ind Microbiol 13: 120–125.
36. Rix U, Zheng JT, Rix LLR, Greenwell L, Yang K, et al. (2004) The dynamic
structure of jadomycin B and the amino acid incorporation step of its
biosynthesis. J Am Chem Soc 126: 4496–4497.
37. Jakeman DL, Farrell S, Young W, Doucet RJ, Timmons SC (2005) Novel
jadomycins: incorporation of non-natural and natural amino acids. Bioorg Med
Chem Lett 15: 1447–1449.
38. Zhang HT, Zhang W, Jin Y, Jin MF, Yu XJ (2008) A comparative study on the
phylogenetic diversity of culturable actinobacteria isolated from five marine
sponge species. Antonie van Leeuwenhoek 93: 241–248.
39. Williams PG, Buchanan GO, Feling RH, Kauffman CA, Jensen PR, et al. (2005)
New cytotoxic salinosporamides from the marine actinomycete Salinispora tropica.
J Org Chem 70: 6196–6203.
40. Fiedler HP, Bruntner C, Riedlinger J, Bull AT, Knutsen G, et al. (2008)
Proximicin A, B and C, novel aminofuran antibiotic and anticancer compounds
isolated from marine strains of the actinomycete Verrucosispora. J Antibiot 61:
158–163.
41. Romero F, Espliego F, Baz JP, De Quesada TG, Gravalos D, et al. (1997)
Thiocoraline, a new depsipeptide with antitumor activity produced by a marine
Micromonospora.1. Taxonomy, fermentation, isolation, and biological activities.
J Antibiot 50: 734–737.
42. Baz JP, Canedo LM, Puentes JLF, Elipe MVS (1997) Thiocoraline, a novel
depsipeptide with antitumor activity produced by a marine Micromonospora.2.
Physico-chemical properties and structure determination. J Antibiot 50: 738–
741.
43. Charan RD, Schlingmann G, Janso J, Bernan V, Feng XD, et al. (2004)
Diazepinomicin, a new antimicrobial alkaloid from a marine Micromonospora sp..
J Nat Prod 67: 1431–1433.
44. Padgitt PJ, Moshier SE (1987) Mycobacterium poriferae sp. nov., a scotochromo-
genic, rapidly growing species isolated from a marine sponge. Int J Syst Bacteriol
37: 186–191.
45. Izumi H, Gauthier ME, Degnan BM, Ng YK, Hewavitharana AK, et al. (2010)
Diversity of Mycobacterium species from marine sponges and their sensitivity to
antagonism by sponge-derived rifamycin-synthesizing actinobacterium in the
genus Salinispora. FEMS Microbiol Lett 313: 33–40.
46. Hirsch AM, Valde ´s M (2010) Micromonospora: An important microbe for
biomedicine and potentially for biocontrol and biofuels. Soil Biol Biochem 42:
536–542.
47. Lampert Y, Kelman D, Nitzan Y, Dubinsky Z, Behar A, et al. (2008)
Phylogenetic diversity of bacteria associated with the mucus of Red Sea corals.
FEMS Microbiol Ecol 64: 187–198.
Coral Actinomycetes
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