Antonie van Leeuwenhoek (2014) 106:623–635

DOI 10.1007/s10482-014-0233-1

ORIGINAL PAPER

Detection of polyketide synthase and nonribosomal peptide

synthetase biosynthetic genes from antimicrobial coral-
associated actinomycetes
Jie Li • Jun-De Dong • Jian Yang •

Xiong-Ming Luo • Si Zhang

Received: 1 May 2014 / Accepted: 6 July 2014 / Published online: 5 September 2014
Ó Springer International Publishing Switzerland 2014

Abstract The diversity and properties of actinobac- PKS-I and four NRPS fragments showed \70 %
teria, predominant residents in coral holobionts, have similarity to their closest relatives, which suggested
been rarely documented. In this study, we aimed to the novelty of these genes. This study helps uncover
explore the species diversity, antimicrobial activities the genetic capacity of stony coral-associated actino-
and biosynthetic potential of culturable actinomycetes mycetes to produce bioactive molecules.
within the tissues of the scleractinian corals Porites
lutea, Galaxea fascicularis and Acropora millepora Keywords Scleractinian coral  Actinomycetes 
from the South China Sea. A total of 70 strains Antimicrobial activity  PKS  NRPS
representing 13 families and 15 genera of actinobac-
teria were isolated. The antimicrobial activity and
biosynthetic potential of fifteen representative fila-
mentous actinomycetes were estimated. Crude fer- Introduction
mentation extracts of 6 strains exhibited comparable
or greater activities against Vibrio alginolyticus than A large and diverse bacterial population associate with
ciprofloxacin. Seven of the 15 actinomycetes strains coral mucus, tissues and skeleton (Rosenberg et al.
possess type I polyketide synthases (PKS-I) and/or 2007). An abundance of actinobacteria has been
nonribosomal peptide synthetases (NRPS) genes. Nine shown to be an important part of the bacteria
tested strains possess type II polyketide synthases community associated with scleractinian corals (Lam-
(PKS-II). Phylogenetic analysis based on 16S rRNA pert et al. 2006; Thurber et al. 2009; Barott et al. 2011;
gene sequences indicated that these PKS and NRPS Nithyanand et al. 2011). At present, there are only a
gene screening positive strains belong to genera few reports focusing on isolation of actinobacteria
Nocardiopsis, Pseudonocardia, Streptomyces, Micro- from scleractinian corals (Nithyanand et al. 2011;
monospora, Amycolatopsis and Prauserella. One Yang et al. 2013). Until now, pure isolates of
scleractinian coral-associated Actinomycetales were
mainly distributed in the genera Kocuria, Micrococcus
J. Li  J.-D. Dong  J. Yang  X.-M. Luo  S. Zhang (&)
CAS Key Laboratory of Tropical Marine Bio-resources (Lampert et al. 2006; Yang et al. 2013), Dermatophi-
and Ecology, RNAM Center for Marine Microbiology, lus, Kytococcus (Lampert et al. 2006), Janibacter
Guangdong Key Laboratory of Marine Materia Medica, (Kageyama et al. 2007), Brachybacterium, Brevibac-
South China Sea Institute of Oceanology, Chinese
terium (Nithyanand and Pandian 2009; Yang et al.
Academy of Sciences, 164 West Xingang Road,
Guangzhou 510301, People’s Republic China 2013), Arthrobacter (Shnit-Orland and Kushmaro
e-mail: [email protected] 2009), Corynebacterium (Ben-Dov et al. 2009),

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Streptomyces, Propionibacterium (Nithyanand et al. Coral reefs are widely distributed across the South
2010), Curtobacterium (Nithyanand et al. 2011), China Sea, with a total reef area of approximately
Leucobacter, Microbacterium (Cardenas et al. 2012), 7,974 km2, matching the Great Barrier Reef in size,
Rothia (Chiu et al. 2012), Dietzia and Micromonos- latitudinal range and biodiversity (Liu et al. 2009). The
pora (Yang et al. 2013). Among them, there are only Luhuitou fringing reef located in Sanya, southern
two mycelium-forming actinomycetes groups, Strep- Hainan Island, is approximately 3,500 m long and
tomyces and Micromonospora. 250–500 m wide and consists of approximately 70 %
Accumulated evidence suggests that unique niches of the coral species so far reported for Hainan Island
confer marine animal- and plant-associated bacteria and its offshore islands (Liu et al. 2009). However, no
with special properties. The marine invertebrate- research has been carried out for investigation of the
associated bacteria are considered to be a potential actinomycetes associated with the corals located in the
source of natural bioactive compounds of low molec- South China Sea. In this study, the diversity of
ular weight (Kobayashi and Ishibashi 1993; Li 2009). actinomycetes associated with three dominant coral
A previous report indicated that antibiotic production species in the Luhuitou fringing reef, including Porites
protects the macroorganism host by preventing infec- lutea, Galaxea fascicularis and Acropora millepora,
tion with pathogens or fouling with other organisms was investigated using a culture-based approach.
(Rypien et al. 2010). Actinobacteria, a significant Furthermore, the capacities of these coral-associated
source of natural products, are frequent components of strains to produce active antimicrobial compounds
these symbiotic communities. The association of were determined.
actinobacteria with marine sponges has attracted much
attention (Schneemann et al. 2010). In contrast, fewer
studies focused on coral-associated actinobacteria, but Materials and methods
recent reports about the antimicrobial activities of
coral-associated actinobacteria (Nithyanand and Pan- Sample collection
dian 2009; Nithyanand et al. 2011) have captured our
interest. Isolating and screening of coral-associated P. lutea, G. fascicularis and A. millepora samples
actinobacteria will provide a new possible approach were collected from the Luhuitou fringing reef
for discovering valuable bioactive compounds. (18°130 N, 109°280 E) in July 2011 using a punch and
A diverse array of bioactive molecules with appli- hammer. The corals were at a depth of 3–5 m and at a
cations in medicine, agriculture and biochemical temperature of approximately 27–28 °C. The average
research are synthesized by polyketide synthases pH was 8.78 ± 0.01, and the salinity was 34 %. Coral
(PKS) and nonribosomal peptide synthetases (NRPS) fragment surfaces were rinsed with sterile seawater to
(Ayuso-Sacido and Genilloud 2005). Similar to remove loosely attached bacteria and immediately
metabolism in terrestrial microbes, most metabolic placed in Ziploc bags, then were placed on ice until
pathways in marine microbes are mainly derived from processed (within 4 h).
type I polyketide synthases (PKS-I), type II polyketide
synthases (PKS-II), NRPS and PKS-NRPS (Zhang Isolation of actinobacteria from coral samples
et al. 2010). Molecular methods for analyzing and
comparing the genetic variation within these genes are Coral fragments measuring ca. 292 cm were air-
useful for screening potential bioactive molecule brushed with sterile seawater to obtain a tissue slurry.
producing isolates (Metsä-Ketelä et al. 1999; Ayuso- The retained slurry was subjected to a dispersion and
Sacido and Genilloud 2005; Gontang et al. 2010). To differential centrifugation process (Hopkins et al.
the best of our knowledge, there are no reports about 1991). One milliliter suspensions were serially diluted
the functional genes related to secondary metabolism by a factor of 10-2 and 10-3, then spread on isolation
of stony coral-associated actinomycetes. It is signif- agar media Nos. 1 through 5 as follows: 1, marine agar
icant and promising to investigate the stony coral- 2216 (BD; Becton, Dickinson and Company); 2, yeast
associated actinomycetes regarding their diversity as extract agar (yeast extract 0.25 g, K2HPO4 0.5 g, 1
well as their potential for secondary metabolite liter of natural seawater); 3, trehalose proline agar
biosynthesis. [trehalose 5 g, proline 1 g, (NH4)2SO4 1 g,

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MgSO47H2O 1 g, CaCl2 2 g, K2HPO4 1 g, 1 liter of disc assay method (Mearns-Spragg et al. 1998). Five
natural seawater]; 4, raffinose histidine agar (histidine microliter of each suspension was pipetted onto paper
0.1 g, raffinose 1.0 g, Na2HPO4 0.3 g, KCl 1.7 g, discs (6 mm in diameter), and the zones of inhibition
MgSO47H2O 0.05 g, FeSO47H2O 0.01 g, CaCO3 were measured after 24 h incubation at 25 °C. DMSO
0.02 g, 1 liter of natural seawater); and 5, pyruvic acid was used as the negative control, and ciprofloxacin
sodium asparagine agar (pyruvic acid sodium 0.5 g, (20 mg ml-1) was used as the positive control. Each
asparagine 0.1 g, KH2PO4 0.95 g, K2HPO4 0.6 g, treatment was replicated three times.
MgSO47H2O 0.025 g, CaCl22H2O 0.003 g, 1 liter of
natural seawater). All media contained 12 g agar, and PCR amplification of genes involved in secondary
the pH value was 7.5. Petri dishes were incubated at metabolism
28 °C, corresponding to the temperature of the
ambient seawater for 3–4 weeks. Three sets of PCR primers were used: A3F (50 -GCS
TAC SYS ATS TAC ACS TCS GG-30 ) and A7R (50 -
Bacterial identification by 16S rRNA gene SAS GTC VCC SGT SCG GTA S-30 ) targeting NRPS
sequence analysis adenylation domain sequences; K1F (50 -TSA AGT
CSA ACA TCG GBC A-30 ) and M6R (50 -CGC AGG
Purified strains were cultivated on marine agar plates TTS CSG TAC CAG TA-30 ) targeting PKS-I KS and
at 28 °C. Genomic DNA extraction, amplification and methyl malonyl transferase domains (Ayuso-Sacido
16S rRNA gene sequencing were performed as and Genilloud 2005); and KSaF (50 -TSG CST GCT
described previously (Li et al. 2007). Identification TGG AYG CSA TC-30 ) and KSaR (50 -TGG AAN
of phylogenetic neighbors and calculation of pairwise CCG CCG AAB CCG CT-30 ) targeting KSa genes
16S rRNA gene sequence similarities were achieved (Metsä-Ketelä et al. 1999). PCR amplifications were
using the EzTaxon-e server (Kim et al. 2012). performed in a Mastercycler Pro (Eppendorf, Ham-
Phylogenetic trees were generated using the neigh- burg, Germany) in a final volume of 25 ll containing
bor-joining (Saitou and Nei 1987) algorithms with the 0.2 lmol l-1 of each primer, 0.1 mmol l-1 of each of
MEGA package version 5.05 (Tamura et al. 2011) the four dNTPs (TaKaRa), 1 ll of extracted DNA, 0.5
after multiple alignment of the data by CLUSTAL_X unit of Taq DNA polymerase and 12.5 ll of 29GC
(Thompson et al. 1997). The 16S rRNA gene sequence buffer (TaKaRa). PKS-I, PKS-II and NRPS amplifi-
data were deposited in GenBank under accession cations with degenerate primers were performed
numbers JX454450-JX454519. according to the following profiles: 5 min denatur-
ation at 96 °C and 30 cycles of 1 min at 96 °C, 1 min
Fermentation of isolates and analysis at either 57 °C (for K1F/M6R, A3F/A7R) or 58 °C
of fermentation extracts (for KSaF/KSaR) and 1 min extension at 72 °C,
followed by 10 min at 72 °C. The sizes of the
Fermentation was performed in 250-ml Erlenmeyer amplicons were 1,200–1,400 bp (K1F/M6R), 613 bp
flasks containing 50 ml of medium (4 g malt extract, (KSaF/KSaR) and 700–800 bp (A3F/A7R). Amplifi-
4 g yeast extract, 4 g glucose, pH 7.2 in 1,000 ml of cation products were analyzed by electrophoresis in
sea water, pH 7.0) at 28 °C on shaker with 200 rpm. 1 % agarose gels. The correct size amplicons were
After incubated for 7–8 days, the fermentation broth purified using the E.Z.N.A.Ò Gel Extraction Kit
was extracted with 50 ml ethyl acetate. Each ethyl (Omega Bio-Tek), then cloned into pGEM-T Easy
acetate extract was then evaporated under reduced vector (Promega). The positive clones were amplified
pressure and resuspended in dimethyl sulfoxide by PCR using primer pairs T7 (50 -TA-
(DMSO) to a final concentration of 20 mg ml-1. ATACGACTCACTATAGGG-30 ) and SP6 (50 - CAT-
The concentrated extracts were tested for antimicro- ACGATTTAGGTGACACTATAG-30 ). Sequences of
bial activity against reported coral pathogens Vibrio the inserts were determined by an ABI 37309l DNA
coralliilyticus ATCC BAA-450, Vibrio alginolyticus sequencer using T7 and/or SP6 primers.
ATCC 17749 and Vibrio harveyi ATCC 33867 (Ben- Translated protein sequences were derived from
Haim et al. 2003; Rosenberg et al. 2007; Cervino et al. nucleotide sequences using the ORF FINDER (http://
2008). For antimicrobial testing, we used the paper www.ncbi.nlm.nih.gov/projects/gorf/). The deduced

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Table 1 Taxonomic Families Genera Galaxea Porites Acropora

distribution of fascicularis lutea millepora
actinobacteria associated
with scleractinian corals Brevibacteriaceae Brevibacterium 5a 3 2
Dermabacteraceae Brachybacterium 2 2 0
Jiangellaceae Jiangella 3 0 1
Microbacteriaceae Microbacterium 1 0 3
Micrococcaceae Micrococcus 1 0 0
Micromonosporaceae Micromonospora 1 3 3
Mycobacteriaceae Mycobacterium 0 3 0
Nocardiaceae Gordonia 1 4 2
Nocardiopsaceae Nocardiopsis 5 3 3
Promicromonosporaceae Cellulosimicrobium 0 1 2
Pseudonocardiaceae Pseudonocardia 1 0 3
Amycolatopsis 2 0 0
Prauserella 1 0 0
Streptomycetaceae Streptomyces 2 5 1
Tsukamurellaceae Tsukamurella 1 0 0
a
Number of strains Total 26 24 20
isolated from each sample

amino acid sequences of the NRPS and PKS genes Brachybacterium (4 strains, 5.7 %), Jiangella (4
were used as queries to search the related proteins in strains, 5.7 %), Microbacterium (4 strains, 5.7 %),
the NCBI nr protein database using the BLASTP Pseudonocardia (4 strains, 5.7 %), Cellulosimicrobi-
algorithm with the default parameters. Multiple um (3 strains, 4.3 %), Mycobacterium (3 strains,
sequence alignment was performed using CLUSTAL 4.3 %), Amycolatopsis (2 strains, 2.9 %), Micrococ-
X. NRPS and PKS phylogenetic trees were con- cus (1 strain, 1.4 %), Prauserella (1 strain, 1.4 %) and
structed using the MEGA 5.05 package. The neighbor- Tsukamurella (1 strain, 1.4 %) (Table 1; Fig. 1).
joining method was used with 1,000 bootstrap The 16S rRNA gene sequences were compared
resampling. The obtained NRPS and PKS gene with the database of valid species using the EzTaxon-e
sequences were deposited into GenBank under the server. Results showed that several strains have
accession numbers KF470759, KF470761 to relatively low similarities to the type strains of the
KF470771 and KF470773 to KF470783. corresponding valid species. The 16S rRNA gene
sequences of strains SCSIO 11456 and SCSIO 11455
showed 98 % identity to Amycolatopsis echigonensis
Results (AB248535) and Amycolatopsis benzoatilytica
(AY957506). Strains SCSIO 11434, SCSIO 11735
Isolation and phylogenetic analysis and SCSIO 11395 showed 97 % identity to Brevibac-
of actinobacteria terium picturae (AJ620364). 16S rRNA gene
sequence similarity between strain SCSIO 11529 and
A total of 70 strains were isolated from three coral Prauserella marina (FJ444996) was 97 %. Therefore,
tissue samples, including 26 strains from G. fascicu- these strains most likely represent new species, and
laris, 24 strains from P. lutea, and 20 strains from A. taxonomic studies are underway.
millepora. According to identification procedures,
these isolates could be classified into 13 families and Antimicrobial activity of fermentation extracts
15 genera, including Nocardiopsis (11 strains,
15.7 %), Brevibacterium (10 strains, 14.3 %), Strep- Fifteen filamentous actinomycetes were cultivated in
tomyces (8 strains, 11.4 %), Gordonia (7 strains, fermentation broth. Extracts of these cultures were
10.0 %), Micromonospora (7 strains, 10 %), tested against three indicator pathogens, V.

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Fig. 1 Neighbor-joining
tree showing the
phylogenetic relationships
based on 16S rRNA gene
sequences of culturable
actinobacteria associated
with scleractinian corals.
Sequences obtained in this
study are in bold. Bootstrap
values (expressed as
percentages of 1,000
replications) greater than
50 % are given at the nodes.
Bar, 2 nt substitution per 100
nt. Strains marked with a
triangle were isolated from
Acropora millepora; strains
marked with a circle were
isolated from Galaxea
fascicularis; strains marked
with a square were isolated
from Porites lutea

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Table 2 Antimicrobial activities of actinomycetes associated with scleractinian corals

Strain Vibrio Vibrio Vibrio PKS- PKS- NRPS Closest type strain (% identity)
harveyi coralliilyticus alginolyticus I II

SCSIO – – – – ? – Amycolatopsis echigonensis LC2, AB248535

11455 (97.911)
SCSIO – – 0.88 ± 0.03a – – – Jiangella gansuensis YIM 002, AY631071
11438 (99.708)
SCSIO – – – – – – Jiangella alba YIM 61503, FJ157186
11458 (99.863)
SCSIO – 1.70 ± 0.27 2.18 ± 0.25 ? ? – Micromonospora aurantiaca ATCC 27029,
11524 CP002162 (100)
SCSIO – – 1.30 ± 0.17 – – – Nocardiopsis alba DSM 43377, X97883
11520 (100)
SCSIO – – – ? ? ? Nocardiopsis dassonvillei subsp. albirubida
11453 DSM 40465, X97882 (99.725)
SCSIO – – 1.23 ± 0.21 – ? ? Nocardiopsis flavescens SA6, GU997639
11462 (100)
SCSIO – – 0.88 ± 0.08 ? ? ? Nocardiopsis synnemataformans IMMIB
11449 D-1215, Y13593 (99.596)
SCSIO – – 1.77 ± 0.31 – – – Nocardiopsis yanglingensis A18, GQ463465
11460 (99.862)
SCSIO – – – – – – Prauserella marina MS498, FJ444996
11529 (96.990)
SCSIO – – 2.33 ± 0.21 ? – ? Pseudonocardia kongjuensis LM 157,
11457 AJ252833 (99.395)
SCSIO – – 0.85 ± 0.07 – ? ? Streptomyces fimicarius ISP 5322,
11527 AY999784 (100)
SCSIO – – 0.90 ± 0.14 ? ? ? Streptomyces rutgersensis NBRC 12819,
11469 AB184170 (100)
SCSIO – 1.03 ± 0.06 0.97 ± 0.15 ? ? – Streptomyces variabilis NBRC 12825,
11531 AB184884 (99.859)
SCSIO – – 1.23 ± 0.25 ? ? ? Streptomyces viridodiastaticus NBRC 13106,
11717 AB184317 (100)
DMSO – – –
Ciprofloxacin 2.50 ± 0.10 2.63 ± 0.38 1.50 ± 0.10
‘‘–’’indicates negative results; ‘‘?’’indicates positive results
a
diameter of the zones of inhibition (cm)

coralliilyticus, V. alginolyticus and V. harveyi. Eleven antimicrobial activity against V. harveyi. Detailed
isolates exhibited activity against V. alginolyticus. results are reported in Table 2.
Among them, the extracts from strains SCSIO 11460,
SCSIO 11462, SCSIO 11520 and SCSIO 11717 Occurrence and phylogenetic analysis
showed activity comparable to that of ciprofloxacin. of biosynthetic genes
The antibiotic activity of extracts from strains SCSIO
11457 and SCSIO 11524 was greater than that of PCR amplification of genes using the PKS-I specific
ciprofloxacin (Table 2). Moreover, strain SCSIO primers resulted in products of the appropriate size for
11524 also presented a 17 mm inhibition zone of seven of the isolates including 2 Nocardiopsis strains,
against V. coralliilyticus. Strain SCSIO 11531 also 1 Pseudonocardia strain, 3 Streptomyces strains and 1
showed activity against both V. alginolyticus and V. Micromonospora strain (Tables 2, 3). The nucleic acid
coralliilyticus. None of the isolates showed sequences were translated prior to phylogenetic

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analyses. Using GenBank BLASTP search, the PKS-I identified as a product of marine-derived Streptomyces
sequences shared 47–99 % sequence similarity with maritimus (Sitachitta et al. 1996).
their closest matches (Table 3). The sequence from Three Nocardiopsis strains, 1 Pseudonocardia and
Pseudonocardia sp. strain SCSIO 11457 shared only 3 Streptomyces strains possess NRPS genes. NRPS
47 % similarity with its closest neighbor, Streptomy- fragments derived from SCSIO 11469, SCSIO 11449,
ces sp. D22, at the amino acid level. PKS-I sequence SCSIO 11527 and SCSIO 11462 showed 51–70 %
retrieved from SCSIO 11469 was most closely related similarity to their closest relatives (Table 3). This
to a gene product involved in the production of result suggested the novelty of these NRPS genes. We
polyene antibiotic FR-008 (Table 3). In addition, noticed that the NRPS sequences from strains Nocar-
PKS-I sequences from Streptomyces strains SCSIO diopsis sp. SCSIO 11449 and Streptomyces sp. SCSIO
11717 and SCSIO 11531 showed the highest identity 11527 shared the same closest match from Strepto-
to the PKS-I gene possessed by the obligate marine myces marokkonensis and clustered together in the
actinomycetes Salinispora arenicola (Table 3). phylogenetic tree (Table 3; Fig. 3). The NRPS
Three Nocardiopsis strains, 1 Amycolatopsis strain, sequence from SCSIO 11462 (Nocardiopsis sp.)
4 Streptomyces strains and 1 Micromonospora strain showed the highest identity (67 %) to the sequence
showed PKS-II gene screening positive results from the close relative Nocardiopsis alkaliphila,
(Tables 2; 3). The PKS-II sequences shared meanwhile the second-most similar (66 %) amino
79–100 % sequence similarity with their closest acid sequence was from the marine-derived Verruco-
matches (Table 3). The KSa sequences obtained in sispora maris AB-18-032 (Table 3).
this study and a number of the reference sequences
reported by Metsä-Ketelä et al. (2002) and from
GenBank were used in the phylogenetic analysis Discussion
(Fig. 2). The PKS-II sequence from SCSIO 11527
showed the highest similarity (90 %) at the amino acid Although diverse actinobacteria have been detected in
level to Streptomyces argillaceus ATCC 12956, which culture-independent research, cultured actinobacterial
produces anthracycline antibiotics mithramycin resources from stony corals are extremely limited. So
(Lombó et al. 1999). Both of them clustered with the far, only members of the genera Streptomyces,
sequence involved in nogalamycin production Brachybacterium, Brevibacterium, Microbacterium,
(Fig. 2). Both PKS-II sequences from strain SCSIO Micrococcus and Micromonospora have been isolated
11524 and its closest neighbor, Micromonospora sp. from the corals Acropora digitifera (Nithyanand and
L5, grouped with those sequences involved in the Pandian 2009; Nithyanand et al. 2011), Platygyra
production of anthracycline antibiotics, including carnosus (Chiu et al. 2012), Fungia scutaria (Lampert
aclacinomycins and daunorubicin-doxorubicin et al. 2006) and Tubastraea coccinea (Yang et al.
(Grimm et al. 1994; Räty et al. 2002). The closest 2013). This is the first study to report the rarely
neighbor of the PKS sequence from strain SCSIO recovered genera of Actinomycetales, e.g., Mycobac-
11455 derived from an uncultured bacterium. Both terium, Gordonia, Tsukamurella, Jiangella, Cellulos-
these two sequences grouped with the PKS gene imicrobium, Pseudonocardia, Amycolatopsis,
present in the genome of Thermobifida fusca YX Prauserella and Nocardiopsis, isolated from the
(Lykidis et al. 2007). Sequences derived from three tissues of scleractinian corals. A much larger number
Nocardiopsis isolates (SCSIO 11453, SCSIO 11449, and more diverse actinobacteria strains were isolated
SCSIO 11462) did not clearly group with any of the in this study compared with previously reported ones
annotated sequences used for analysis. The PKS (Nithyanand et al. 2011; Yang et al. 2013), in which a
sequences from Streptomyces sp. SCSIO 11717 and single medium, such as starch casein agar, and routine
SCSIO 11531 grouped with those sequences involved serial dilution and plating techniques were applied.
in the production of spore pigments (Davis and Chater This difference highlights the necessity of using
1990; Bergh and Uhlén 1992; Blanco et al. 1993). The various media and methods for isolation of culturable
closest BLAST match (99 % amino acid identity) of coral-associated actinomycetes, especially rare non-
the PKS sequence from isolate SCSIO 11469 was Streptomyces actinomycetes. This result also suggests
related to enterocin production, which was first that diverse unexplored culturable actinobacteria are

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Table 3 Similarity to the closest relatives in GenBank of PKS-I, PKS-II and NRPS amino acid sequences of isolates from stony
corals
Isolate Genus Accession Top BLAST match (NCBI accession no.) Identity (%)
no.

PKS-I
SCSIO 11524 Micromonospora KF470779 Acyl transferase from Micromonospora aurantiaca 99
ATCC 27029 (YP_003835579)
SCSIO 11449 Nocardiopsis KF470778 Acyl transferase from Nocardiopsis dassonvillei 79
subsp. dassonvillei DSM 43111
(YP_003678449.1)
SCSIO 11453 Nocardiopsis KF470781 Hypothetical protein from Nocardiopsis sp. CNS639 85
(WP_019606713)
SCSIO 11457 Pseudonocardia KF470782 Modular polyketide synthase from Streptomyces sp. 47
D22 (ACL97730)
SCSIO 11717 Streptomyces KF470777 Hypothetical protein from Salinispora arenicola 83
(WP_020637975)
SCSIO 11531 Streptomyces KF470780 Hypothetical protein from Salinispora arenicola 77
(WP_020637975)
SCSIO 11469 Streptomyces KF470783 Polyketide synthase from Streptomyces sp. FR-008 91
(AAQ82568)
PKS-II
SCSIO 11455 Amycolatopsis KF470759 Putative type II PKS ketosynthase alpha subunit 98
from uncultured bacterium, soil, Australia
(AGG43855)
Actinorhodin polyketide beta-ketoacyl synthase 79
alpha subunit/3-oxoacyl-ACP synthase I from soil
bacterium Thermobifida fusca YX (YP_289279.1)
SCSIO 11524 Micromonospora KF470763 Beta-ketoacyl synthase from Micromonospora sp. 99
L5 (YP_004084030.1)
SCSIO 11462 Nocardiopsis KF470764 Putative ketoacyl synthase from Nocardiopsis sp. 90
HB383, host ‘‘Halichondria panacea’’
(ADI24420)
SCSIO 11449 Nocardiopsis KF470761 Beta-ketoacyl synthase from Nocardiopsis 99
dassonvillei subsp. dassonvillei DSM 43111
(YP_003682168.1)
SCSIO 11453 Nocardiopsis KF470767 Beta-ketoacyl synthase from Nocardiopsis 98
dassonvillei subsp. dassonvillei DSM 43111
(YP_003682168)
SCSIO 11717 Streptomyces KF470762 Ketosynthase from Streptomyces sp. JS-14, soil 99
sediments from mangrove plantations in Manakudi
estuary near Arabian Sea (ADC67077)
SCSIO 11531 Streptomyces KF470768 Ketosynthase from Streptomyces sp. JS-14, soil 100
sediments from mangrove plantations in Manakudi
estuary near Arabian Sea (ADC67077)
SCSIO 11469 Streptomyces KF470765 Putative keto synthase alpha EncA from marine 99
isolate Streptomyces maritimus (AAF81728)
SCSIO 11527 Streptomyces KF470766 Ketoacyl synthase from Streptomyces argillaceus 90
ATCC 12956 (CAA61989)

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Table 3 continued
Isolate Genus Accession Top BLAST match (NCBI accession no.) Identity (%)
no.

NRPS
SCSIO 11462 Nocardiopsis KF470776 Amino acid adenylation domain from Nocardiopsis 67
alkaliphila, desert soil in Egypt (WP_017606416)
Amino acid adenylation from Verrucosispora maris 66
AB-18-032 (YP_004406681)
SCSIO 11453 Nocardiopsis KF470773 Non-ribosomal peptide synthetase modules from 97
Nocardiopsis synnemataformans
(WP_017563645)
SCSIO 11449 Nocardiopsis KF470771 Non-ribosomal peptide synthetase from 70
Streptomyces marokkonensis OAct362, soil
sample from coastal wetland China: the Yellow
River Delta(AGO59056)
SCSIO 11457 Pseudonocardia KF470774 Amino acid adenylation domain-containing protein 89
from Pseudonocardia sp. P1 (WP_010227760)
SCSIO 11717 Streptomyces KF470769 Hypothetical protein from Streptomyces gancidicus 97
(WP_006129818)
SCSIO 11469 Streptomyces KF470770 Dimodular nonribosomal peptide synthase DhbF 51
from Streptomyces rimosus (WP_003985597)
SCSIO 11527 Streptomyces KF470775 Non-ribosomal peptide synthetase from 69
Streptomyces marokkonensis OAct362, soil
sample from coastal wetland China: the Yellow
River Delta(AGO59056)

associated with scleractinian corals. Therefore, the the extracts from stony corals showed less antibacte-
scleractinian corals are a promising source of novel rial activity, which implies that stony corals reply via
actinobacteria species. other means to combat microbial attachment (Kelman
Actinomycetes are a well-known, rich source of et al. 2009). The PKS and NRPS primers used in this
bioactive agents (Baltz 2008; Bérdy 2012). In the study successfully detected the targeted portions of
recent years since the high rate of re-discovery of these genes in a number of our isolates. Moreover, the
known compounds in terrestrial actinomycetes, acti- prevalence of antimicrobial activities against the coral
nomycetes isolated from the marine environment, pathogens detected in this study and in previous works
such as sediments, sponges and seaweeds, have (Nithyanand and Pandian 2009; Nithyanand et al.
received considerable attention (Lane and Moore 2011) implies that these bioactive actinomycetes may
2011). In the results reported by Sun et al. (2012), 12 contribute to the chemical defense of corals (Shnit-
soft coral Scleronephthya sp.-associated filament Orland and Kushmaro 2009).
actinomycetes belonging to the genera Streptomyces The phylogenetic relationship analysis of the PKS
and Micromonospora were analyzed for ketoacyl- gene product sequences obtained in this study and the
synthase genes in the PKS-II gene cluster and most reference strains reported by Metsä-Ketelä et al. (2002)
(83.3 %) of them possess the KSa gene. In this study, permitted us to infer the gene function of the PKS
KSa gene was detected in 60.0 % of the stony coral sequences identified in the present study. The amplified
associated actinomycetes. They distributed in diverse portion of the PKS-II gene from isolates SCSIO 11524
genera including Nocardiopsis, Amycolatopsis, Strep- and SCSIO 11527 grouped in the anthracycline clades.
tomyces and Micromonospora. Moreover, most This result suggested that these two isolates may
(66.7 %) of the screened actinomycetes were tested demonstrate the potential to produce molecules related
positive for at least one biosynthetic pathway. In to this class of antitumor antibiotics. The PKS-II
comparison with extracts from soft corals or sponges, sequences from isolates SCSIO 11453, SCSIO 11449,

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632 Antonie van Leeuwenhoek (2014) 106:623–635

100 SCSIO 11524 (KF470763)

53 Micromonospora sp. L5 Beta-ketoacyl synthase (YP_004084030.1)
86 Streptomyces galilaeus ATCC31615 aclacinomycins (AF257324) Anthracycline
58
Streptomyces peucetius ATCC 29050 daunorubicin-doxorubicin (L35560) Anthracycline
Streptomyces rimosus oxytetracycline (Z25538) Tetracycline
Streptomyces nogalater ATCC 27451 nogalamycin (AJ224512)
SCSIO 11527 (KF470766) Anthracycline
96 Streptomyces argillaceus ATCC 12956 mithramycin (CAA61989)
99 SCSIO 11455 (KF470759)
81 Uncultured bacterium clone A32_42 (AGG43855)
Thermobifida fusca YX actinorhodin (YP_289279.1)
58 Streptomyces griseus K-63 griseusin (X77865) Naphthoquinone
Streptomyces cinnamonensis A3823.5 monensin (Z11511)
92
97 Streptomyces venezuelae ATCC 10712 jadomycin (AF126429) Angucycline
55 Streptomyces cyanogenus S136 landomycin (AF080235)
Streptomyces glaucescens tetracenomycin (M80674) Tetracenomycin
78 SCSIO 11453 (KF470767)
99 Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111 (YP_003682168.1)
100 SCSIO 11449K (KF470761)
99
SCSIO 11462 (KF470764)
Nocardiopsis sp. HB383 (ADI24420)
98 Streptomyces coelicolor A3(2) Spore colour (X55942)
70
Streptomyces halstedii ORF1 putative ketoacyl synthase (L05390)
93 Streptomyces curacoi ATCC 13385 (M33704)
Spore Pigments
Streptomyces sp. JS-14 ketosynthase gene (ADC67077)
81 SCSIO 11717 (KF470762)
67 SCSIO 11531 (KF470768)
Streptomyces roseofulvus frenolicin (L26338)
Streptomyces coelicolor A3(2) actinorhodin (X63449)
Streptomyces arenae DSM40737 naphthocyclinone (AF098965) Naphthoquinone
96
100 Streptomyces violaceoruber Tu22 granaticin (X16144)
94 Streptomyces antibioticus ATCC11891 (CAC05671)
SCSIO 11469 (KF470765)
100 Streptomyces maritimus putative keto synthase alpha EncA enterocin (AAF81728)
Escherichia coli beta-ketoacyl-ACP synthase I (M24427)

0.1

Fig. 2 Neighbor-joining tree of KSa amino acid sequences of are indicated on the right. The scale bar indicates 0.1 substitutions
isolates from this study (shown in bold) and reference sequences of that occur per site. Percentage bootstrap values of neighbor-joining
known representative PKS-II sequences. Predicted chemical classes analysis from 1,000 resamplings are indicated at the nodes

SCSIO 11462 and SCSIO 11455 just formed a distinct on PKS- and NRPS-targeted PCR amplification and
clade with their closest BLAST matches (90 and 99 % sequencing, there appears to be the possibility of novel
amino acid identity), but not grouped with any of the bioactive molecule production in some of the isolates
described functional chemical classes. Thus, it is obtained in this study. The detection of highly similar
reasonable to think that this group of isolates has the PKS and NRPS sequences in distantly related strains
potential to produce a structurally novel class of suggests that they have undergone a recent horizontal
compounds. Most of the NRPS amino acid sequences gene transfer (HGT) event, which has also been
obtained in this study shared low homology (51–70 %) observed in previous work (Kim et al. 2006; Jensen
with sequences in the GenBank database. Therefore, it et al. 2007; Gontang et al. 2010).
is not easy to speculate on the bioactive metabolites of Most strains, except SCSIO 11453 and SCSIO
these gene products because of the very low amino acid 11455, that tested positive in the genetic prescreening
identities. We were also unable to readily group the approach showed antimicrobial activities to tested
PKS-I amino acid sequences into functional chemical bacteria. Although the NRPS and PKS gene fragments
classes, except for the PKS-I sequence derived from were not detected in strains SCSIO 11438, SCSIO
strain SCSIO 11469, which was closely related to the 11460 and SCSIO 11520, the antimicrobial activity
PKS-I sequence of Streptomyces sp., the polyene was present. This result may imply that additional
antibiotic FR-008 producer (Chen et al. 2003). Based types of bioactive agents or mechanisms are employed

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Antonie van Leeuwenhoek (2014) 106:623–635 633

100 SCSIO 11453 (KF470773)

100 Nocardiopsis synnemataformans NRPS (WP 017563645)
SCSIO 11457 (KF470774)
64 100 Pseudonocardia sp. P1 amino acid adenylation domain-containing protein (WP_010227760.1)
Rhodococcus jostii RHA1 NRPS (YP_702176)
61 SCSIO 11717 (KF470769)
100 Streptomyces gancidicus NRPS (WP_006129818)
100 SCSIO 11469 (KF470770)
Streptomyces rimosus NRPS (WP_003985597)
Streptomyces virginiae NRPS for virginiamycin S (BAF50711)
SCSIO 11462 (KF470776)
80
100 Verrucosispora maris AB-18-032 amino acid adenylation (YP_004406681)
Nocardiopsis alkaliphila amino acid adenylation domain (WP_017606416)
Salinispora tropica CNB-440 amino acid adenylation domain-containing protein (YP_001159640)
Streptomyces coelicolor A3(2) NRPS (NP_631722)
100
Streptomyces marokkonensis OAct362 NRPS (AGO59056)
84
100 SCSIO 11449 (KF470771)
100 SCSIO 11527 (KF470775)

0.1

Fig. 3 Neighbor-joining tree of NRPS amino acid sequences of isolates from this study (shown in bold) and reference sequences. The
scale bar indicates 0.1 substitutions that occur per site. Percentage bootstrap values of neighbor-joining analysis from 1,000
resamplings are indicated at the nodes

in the generation of antimicrobial activities for certain Sequences in major taxonomic groups. Microb Ecol
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