Diversity of Polyketide Synthase (PKS) Genes in Metagenomic Community of Freshwater Sponge Lubomirskia baicalensis
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ISSN 00268933, Molecular Biology, 2012, Vol. 46, No. 6, pp. 790–795. © Pleiades Publishing, Inc., 2012.
Original Russian Text © O.V. Kaluzhnaya, N.V. Kulakova, V.B. Itskovich, 2012, published in Molekulyarnaya Biologiya, 2012, Vol. 46, No. 6, pp. 887–893.
GENOMICS.
TRANSCRIPTOMICS
UDC 577.151.645
Diversity of Polyketide Synthase (PKS) Genes in Metagenomic
Community of Freshwater Sponge Lubomirskia baicalensis
O. V. Kaluzhnaya, N. V. Kulakova, and V. B. Itskovich
Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033;
email: xsun77@rambler.ru
Received March 26, 2012; in final form, April 19, 2012
Abstract—The screening of metagenomic DNA of the microbial community associated with the Baikalian
sponge Lubomirskia baicalensis was performed in order to investigate the presence of polyketide synthase
(PKS) genes. PKS enzyme systems take part in the synthesis of a great number of biologically active sub
stances. The cloning and sequencing of amplified products of the ketosynthase domain section of the PKS
gene cluster revealed 15 fragments of PKS genes with amino acid sequences differing from each other by 35–
65%. A BLASTX analysis showed that all of these sequences belong to KS domains identified in various
groups of microorganisms, i.e., Alpha, Beta, and Deltaproteobacteria; Verrucomicrobia; Cyanobacteria;
and Chlorophyta. Some sequences were related to genes that participate in the biosynthesis of curacin A
(CurI, CurJ), stigmatellin (StiC, StiG), nostophycin (NpnB), and cryptophycin (CrpB). The homology of the
found sequences with those of the EMBL database lies in the range of 50–82%, which indicates that the
freshwater sponge community contains genes that encode new, not yet studied polyketide substances of
potential biotechnological significance.
DOI: 10.1134/S002689331206009X
Keywords: polyketide synthase (PKS) genes, Lubomirskia baicalensis, community of microorganisms,
metagenomic DNA, phylogenetic analysis
INTRODUCTION epothilone), immune suppressors (cyclosporin and
Natural microbial communities constitute an rapamycin), and many others [13]. Polyketides are
important resource of novel antibiotics and other bio synthesized by multienzyme complexes, i.e.,
logically active metabolites. Currently, approximately polyketide synthases (PKS). This is a large family of
50000 different biologically active molecules of multifunctional proteins that use acyl coenzyme A
microbial origin are known [1]. However, only a small monomers as their substrate [14]. Depending on the
portion of all microbial species (0.1–1%) can be cul protein structure and the catalytic mechanisms, three
tured [2]. For this reason, nonculturable microorgan PKS types are recognized. Type I PKSs, which are also
isms must be the most promising potential source of called modular PKSs, contain several functional
novel compounds of pharmaceutical and biotechno domains that participate in the stepwise polyketide
logical importance. Symbiotic communities, in par synthesis. Each module is responsible for one cycle of
ticular sponge communities (phylum Porifera) have chain elongation. Type II PKSs are complexes of sev
long been extensively investigated in this respect [3– eral mono or bifunctional enzymes that act in an iter
9]. As sessile filter feeders, sponges form a close asso ative manner [2]. In these systems, each catalytic
ciation with a wide range of different microorganisms, domain is located in a separate protein; type II PKSs
such as archaea, heterotrophic bacteria, cyanobacte are usually involved in the synthesis of bacterial aro
ria, algae, dinoflagellates, and fungi [10–12]. Bacteria matic antibiotics. Type III PKSs are homodimeric
constitute up to 40% of the biomass of some marine enzymes responsible for the diversity of flavinoids and
sponges; they are currently classified in 25 different differ from the first two types in that they utilize malo
phyla [13]. nyl coenzyme A as their substrate [15, 16].
A considerable amount of structurally diverse sec The biosynthesis of secondary metabolites often
ondary metabolites are polyketides. The number of involves the combined activity of a PKS system and
polyketides of pharmaceutical importance includes structurally similar nonribosomal peptidase system
antibiotics (e.g., gramicidin, erythromycin, and tetra (NRPS). The number and order of modules in the
cycline), anticancer agents (bleomycin and resulting hybrid NRPS–PKS complexes determines
Abbreviations: PKS, polyketide synthase; KS, ketosynthase
the structural diversity of resulting peptide or
domain; NRPS, nonribosomal peptidase system; BAC, biologi polyketide products, and numerous variants of mod
cally active compound. ule combinations determine the diversity of metabo
790DIVERSITY OF POLYKETIDE SYNTHASE (PKS) GENES 791
lites and their biological activity [17]. Actinobacteria, PKS gene fragments obtained were deposited in the
proteobacteria, and cyanobacteria usually contain GenBank database with accession numbers JQ771596
type I enzymes [18]. Each module of a PKS I system to JQ771610.
comprises at least three domains: ketosynthase (КS),
acyltransferase (AT), and acyl carrier protein (AСР).
Since the sequences of PKS modules correspond to RESULTS AND DISCUSSION
gene clusters in microbial genomes, the ability of Polyketide synthases are involved in the biosynthe
microbial communities and individual strains to pro sis of a large number of biologically active microbial
duce biologically active compounds (BACs) can be metabolites. To search for PKS genes in the noncul
analyzed by PCR. Although the biotechnological turable microbial community of the freshwater sponge
potential of the marine sponge microorganisms have L. baicalensis, we used degenerate primers specifically
been extensively studied, little is known in this respect annealing to conserved regions of the ketosynthase
concerning freshwater sponges. However, it is known domain of PKS [16]. Amplification products of
that bacterial strains obtained from unusual and approximately 700 bp were cloned, and inserts were
poorly studied communities often turn out to be a rich sequenced for 85 randomly selected clones. Nucle
source of novel BACs [19]. otide sequences showing less than 97% homology were
We have previously shown that the microbial com considered to be unique. Altogether, 15 different
munity associated with the endemic Baikalian sponge amplicons with 35–65% homology to each other were
Lubomirskia baicalensis is highly diverse and includes identified, all of which belonged to type I modular
microbial groups, such as Actinobacteria, α and PKSs. A BLASTX search was performed to find the
βproteobacteria, Verrucomicrobia, Cyanobacteria, closest known homologues and determine the level of
Bacteroidetes, and Nitrospirae [20]. Since many homology of the amino acid sequence (table). The
marine species of these groups are known producers of newly obtained sequences showed 50–82% homology
bioactive metabolites, it is reasonable to search and to those published previously; for most of them (10 out
comparatively analyze PKS genes in the community of of 15 sequences), the homology level was below 70%.
an endemic Baikalian sponge. This suggests that the genomes of microorganisms
This study is the first to investigate the diversity of associated with the Baikalian sponge contain new, still
PKS genes in the metagenomic community of fresh undescribed PKS genes. A phylogenetic analysis was
water sponge L. baicalensis. performed to classify the identified sequences by
enzyme groups. For this purpose, type I modular PKS
sequences with the highest homology to the new KS
EXPERIMENTAL gene domains were retrieved from the EMBL data
Sponge specimens were collected at a depth of base. PKS I of the filamentous fungus Gibberella mon
15 m during field research in the neighborhood of iformis was used as an outgroup.
Listvyanka (southwestern coast of Lake Baikal) in It was found that KS domain sequences of the
February 2009. Specimens were placed in 70% ethanol microbial community of L. baicalensis can be grouped
and stored at 4°C. Total DNA was isolated using a with known PKSs of the bacterial phyla Proteobacte
RiboSorb kit (Russia) as recommended by the manu ria (the classes of α, β, and δProteobacteria), Ver
facturer. Fragments of KS domains of PKS genes were rucomicrobia, Cyanobacteria, and Acidobacteria, as
amplified using degenerate oligonucleotide primers well as with those of green algae of the order Chloro
DKF (5'GTGCCGGTNCCRTGNGYYTC3') and phyta (figure).
DKR (5'GCGATGGAYCCNCARCARYG3') [16]. A phylogenetic analysis showed that 6 of the 15 clones
The PCR protocol included activation at 95°С for (1aKSLb, 1cKSLb, 1fKSLb, 2aKSLb, 2bKSLb,
5 min, 35 cycles of denaturation at 95°С for 45 s, 3dKSLb, and 5bKSLb) formed a separate cluster
primer annealing at 62°С for 60 s, and elongation at together with the sequences of hybrid NRPS/PKS
72°С for 90 s; and final elongation for 10 min at 72°С. complexes. It is known that, due to their combined
PCR products were cloned in a pTZ57R/T vector activity, NRPS/PKS hybrid systems perform the bio
(Fermentas) and used to transform chemically com synthesis of a number of bioactive compounds, includ
petent E. coli XL1BL cells. Sequences of recombinant ing those of industrial importance (such as rifampicin,
clones that carry an insert of the expected size myxothiazol, curacin, jamaicamide, and others [14]).
(approximately 700 bp) were determined using a CEQ In agreement with this notion, among sequences that
8800 automated sequencer (Beckman Coulter Inc., are phylogenetically close to the new KS domains, the
United States). The sequences were aligned and their tree included the genes of stigmatellin (StiC and StiG),
homology was evaluated using the ClustalW module of curacin (CurI and CurJ), nostophycin (NpnB), and
BioEdit 7.0 software package [21]. A comparison with cryptophycin (CrpB) (figure). Clones of this cluster
the available data was performed using the BLASTX had 53–65% homology of the amino acid sequence; in
program at the NCBI site [22]. Phylogenetic tree was the tree, they clusterized with genes derived from spe
constructed by neighborjoining method (NJ) using cies of different bacterial phyla (figure). This wide
Mega 4 software [23]. Nucleotide sequences of the range of homology possibly indicates the functional
MOLECULAR BIOLOGY Vol. 46 No. 6 2012792 KALUZHNAYA et al.
Homology of KS sequences studied to known PKS genes
BLAST results
Sequence (GenBank Acc. No.)
Closest homologues (GenBank Acc. No.) Homology, %
1aKSLb (JQ771596) Volvox carteri (XP_002951836) 50
1cKSLb (JQ771597) Scytonema hofmanni PCC7110 (AAW55365) 74
1dKSLb (JQ771598) Methylobacterium extorquens DSM 13060 (EHP84300) 66
1fKSLb (JQ771599) uncultured bacteria from East China Sea (ABG20983) 82
2aKSLb (JQ771600) Chlorella variabilis (EFN51779) 64
2bKSLb (JQ771601) Pleurocapsa sp. PCC 7319 (AAW55384) 66
2nKSLb (JQ771602) Myxococcus fulvus HW1 (YP_004664823) 68
3bKSLb (JQ771603) Synechococcus sp. CC9311 (YP_729393) 78
3dKSLb (JQ771604) Chondromyces crocatus (CAQ18829) 67
3hKSLb (JQ771605) Azoarcus sp. KH32C (BAL22601) 76
4aKSLb (JQ771606) Synechococcus sp. CC9311 (YP_729393) 77
4gKSLb (JQ771607) uncultured bacterium, soil sample (ADD65251) 64
5bKSLb (JQ771608) Opitutus terrae PB901 (YP_001818846) 66
5cKSLb (JQ771609) Granulicella mallensis MP5ACTX8 (YP_005058602) 67
5dKSLb (JQ771610) Haliangium ochraceum DSM 14365 (YP_003265646) 61
diversity of BACs synthesized with help of the newly [14, 28, 29]. Thus, bacteria of the order Myxobacteri
detected genes. For sequences retrieved from Gen ales are the most widely used antibiotic producers after
Bank, the highest levels of homology to novel Actinobacteria and fungi [28]. On the other hand,
sequences ranged from 61 to 82% (table). cyanobacteria, which are members of symbiotic com
With a high bootstrap support, the 1cKSLb munities of both marine and freshwater sponges
sequence was joined to a KS domain from the freshwa [20, 30, 31], are a rich source of different toxins [16].
ter cyanobacterium Scytonema hofmanni PCC 7110 1aKSLb and 2aKSLb clones belonged to the
and the stigmatellin gene sequence (StiC, electron same cluster as KS domains of eukaryotic green algae
transport inhibitor) of the soil myxobacterium Stigma Volvox carteri and Chlorella variabilis (order Chloro
tella aurantiaca [24]. This group also included the bio
phyta); their homology to each other was only 48%. At
synthesis genes of known cyanobacterial metabolites,
including curacin (an antitumor toxin, CurI, Lyngbya the same time, the sequence of 1aKSLb had 50%
majuscule) [25], nostophycin (a cytotoxin) [26], and homology to the KS domain of Volvox carteri, while
cryptophycin (a tubulindepolymerizing agent) [27]. 2aKSLb had 64% homology to the respective
The closest homologue of 5bKSLb was a sequence sequence of Chlorella variabilis (table). Interestingly,
from the Opitutus terrae strain PB901 of the phylum this group also included a KS domain sequence from a
Verrucomicrobia. Clone 2bKSLb was grouped with nonculturable symbiont of the marine sponge Disco
KS domains from cyanobacteria Nostoc punctiforme dermia dissolute. It was previously shown that a num
PCC 73102, Pleurocapsa sp. PCC 7319, and Microcys ber of microbial species associated with this sponge are
tis aeruginosa NPCD1, as well as with the CurJ gene of a potential source of different natural compounds with
curacin biosynthesis from Lyngbya majuscula; clone cytotoxic, antibacterial, antifungal, antiviral, and
5dKSLb was grouped with a myxobacterium other bioactive properties [32]. Handa et al. demon
Haliangium ochraceum DSM 14365; clone 1fKSLb strated the presence of green algae, predominantly
was grouped with a nonculturable soil bacterium, Chlorella species, in communities of Baikalian
while 3dKSLb was grouped with δProteobacteria sponges [33]. They also pointed out that the large
Chondromyces crocatus and Stigmatella aurantiaca. number of Baikalian freshwater sponges symbiotic
Thus, this cluster predominantly contained sequences with chlorophyllcontaining microorganisms indi
from different Cyanobacteria species, as well as from cates their importance for the primary product accu
species of the order Myxobacteriales (class δProteo mulation in the lake ecosystem [33]. Green algae are
bacteria of the phylum Proteobacteria). These bacte also fairly common in communities of marine sponges
rial groups have long been extensively studied due to [7, 34], where they are usually present in the outer
their high potential as bioactive metabolite producers layer of the sponge body [35]. Apparently, in freshwa
MOLECULAR BIOLOGY Vol. 46 No. 6 2012DIVERSITY OF POLYKETIDE SYNTHASE (PKS) GENES 793
60
1cKSLb
Scytonema hofmanni PCC 7110, AAW55365 [Cyano]
73 79 StiC Stigmatella aurantiaca, CAD19087 [DeltaP]
Curl Lyngbya majuscula, AAT70107 [Cyano]
67 NpnB Nostoc sp. 152, AEU11006 [Cyano]
65 CrpB Nostoc sp. ATCC 53789, AMB21570 [Cyano]
50 5bKSLb
Opitutus terrae PB901, YP_001818846 [Verruco]
CurJ Lyngbya majuscula, AAT70105 [Cyano]
2bKSLb
51
Pleurocapsa sp. PCC 7319, AAW55384 [Cyano]
NRPS/PKS
88 hybrid
Nostoc punctiforme PCC, YP_001865644 [Cyano]
50 Microcystis aeruginosa, ACF24471 [Cyano] complexes
uncultured bacterium (mangrove soil), ACC99569
5dKSLb
89 Haliangium ochraceum DSM 14365, YP_00326564 [DeltaP]
3dKSLb
Chondromyces crocatus Cmc5, CAQ18829 [DeltaP]
71 StiG Stigmatella aurantiaca Sg a15, CAD19091 [DeltaP]
Nodularia harveyana, AAX44144 [Cyano]
44 94 100 1fKSLb
uncultured bacterium (East China Sea, cost soil), ABG20983
74 1aKSLb
100 Volvox carteri XP_00295183 [Chloroph]
60 PKS I
100 2aKSLb
Chlorella variabilis NC64A, EFN51779 Chlorophyta
55
uncultured bacterial symbiont of sponge Discodermia dissoluta, A
51
2nKSLb
58 Myxococcus fulvus HW1, YP_004664823 [DeltaP]
42 5cKSLb
Granulicella mallensis MP5ACTX8, AEU37572 [Acido]
52 54 Haliangium tepidum, BAG69128 [DeltaP]
91 3bKSLb
100 4aKSLb
54 Synechococcus sp. CC9311, YP_729393 [Cyano]
71 100 Stanieria sp. PCC 7302, AAW55404[Cyano] PKS I
63 Pleurocapsa sp. PCC 7319, AAW55408 [Cyano] Cyanobacteria
uncultured bacteria of rhizosphere soil, ADD65273
80 Scytonema hofmanni PCC 7110, AAW55402[Cyano]
4gKSLb
91 uncultured bacterium of rhizosphere soil, ADD65251
78 Verrucomicrobium spinosum DSM 4136, ZP_02927635 [Verruco]
100 1dKSLb
100 WcbR, RkpAlike Methylobacterium extorquens, EHP84300 [AlphaP]
Sorangium cellulosum, ABD17673 [DeltaP]
92 3hKSLb
100
Azoarcus sp. KH32C, BAL22601 [BetaP] wcbR genes
62 uncultured bacterium of rhizosphere soil, ADD65280 of Proteobacteria
67 79 WcbR Nitrosomonas europaea ATCC 19718, NP_841435 [BetaP]
uncultured marine sponge symbiont, AAX62315
55 100 WcbR Ralstonia sp. 5_7_47FAA, ZP_07677262 [BetaP]
Burkholderia sp. H160, ZP_03267586 [BetaP]
Gibberella moniliformis, PKS I, AAR92209 [Fungi]
0.05
Phylogenetic tree constructed by neighborjoining (NJ) based on fragments of PKS amino acid sequences of the bacterial com
munity of L. baicalensis (240 amino acids). Sequences obtained in this study are designated with filled circles; accession numbers
are given for previously published sequences. Numbers at the tree nodes are bootstrap support values. Evolutionary distance scale
corresponds to five substitutions per 100 bp.
ter communities, eukaryotic microorganisms can also tic is that it also includes WcbR genes, which are
be a source of bioactive substances. involved in the formation of a bacterial capsule [36].
With a high level of support, the tree contains a All sequences of this group represent different classes
clade including sequences 1dKSLb and 3hKSLb, of the phylum Proteobacteria (figure). For instance,
which have 52% similarity; an important characteris the closest homologue of the 1dKSLb sequence is a
MOLECULAR BIOLOGY Vol. 46 No. 6 2012794 KALUZHNAYA et al.
KS domain from Methylobacterium extorquens, a ter and marine communities, as well as thermal spring
methylotroph of the class αProteobacteria (66% sim and soil microbial communities; there are also known
ilarity). This group also contains a sequence from Sor endosymbionts of nematodes and protozoa [43].
angium cellulosum, which belongs to the class δproteo The results of our work show that the microbial
bacteria (order Myxococcales) and is used as a producer community of the freshwater sponge L. baicalensis
of epothilones, which are anticancer compounds [37]. contains numerous PKS genes that belong to microor
Clone 3hKSLb belonged to a group of related ganisms of different systematic groups. It should be
sequences that also included KS domains from noted that many strains that are identified as carrying
βroteobacteria, as well from a nonculturable soil bac homologous genes and used in the phylogenetic anal
terium and a nonculturable sponge symbiont. At the ysis are actually collection strains whose genomes have
level of the amino acid sequence, the highest degree of been sequenced and published. At the same time, a
similarity (76%) was with the KS domain from a soil number of phylogenetically close PKS sequences
bacterium Azoarcus sp. KH32C. Species of this genus belong to bacteria that produce bioactive metabolites
are known as nitrogenfixing plant endosymbionts, as of industrial significance. However, the level of homol
well as bacteria that degrade different aromatic com ogy to known sequences not high enough to imply that
pounds [38]. We previously showed the presence of α similar metabolites were synthesized in the commu
and βProteobacteria in the nonculturable microbial nity of the Baikalian sponge. On the other hand, this
community of L. baicalensis by an analysis of the 16S may suggest that the metagenomic community of
rRNA gene diversity; moreover, the bacteria of these L. baicalensis contains new PKS genes that belong to
two classes made up 35% of the total community [20]. still undescribed microorganisms, as well as that the
Our previous study did not detect any species of the sponge community can produce still unknown biolog
class δproteobacteria; however, the results of this ically active compounds. Our data show the prospects
work suggest that they are probably present in the of further investigations that search for novel bioactive
freshwater sponge community. compounds in bacterial communities of freshwater
3bKSLb and 4aKSLb clones were joined in a tree sponges.
with PKS sequences from different strains of cyano
bacteria (figure). The highest level of KS sequence ACKNOWLEDGMENTS
homology was observed between these two clones
(84%), as well as between them and Synechococcus sp. The study was supported by the Russian Founda
CC9311 (78 and 77%, respectively) (table). Species of tion for Basic Research (project no. 110400323) and
the genus Synechococcus dominate in the pikoplank the Lavrent’ev Foundation of the Siberian Branch of
ton of Lake Baikal [39]; they are also present in the the Russian Academy of Sciences (grant no. 6).
metagenomic community of L. baicalensis [20] and,
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