A bacterial source for mollusk pyrone polyketides

Article abstract of DOI:10.1016/j.chembiol.2012.10.019

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.

Full text of DOI:10.1016/j.chembiol.2012.10.019

Chemistry & Biology
Article
A Bacterial Source for Mollusk Pyrone Polyketides
Zhenjian Lin,1 Joshua P. Torres,6 Mary Anne Ammon,6 Lenny Marett,1,2 Russell W. Teichert,3 Christopher A. Reilly,4
Jason C. Kwan,1 Ronald W. Hughen,2 Malem Flores,6 Ma. Diarey Tianero,1 Olivier Peraud,1 James E. Cox,5 Alan R. Light,2
Aaron Joseph L. Villaraza,7 Margo G. Haygood,8 Gisela P. Concepcion,6 Baldomero M. Olivera,3 and Eric W. Schmidt1,3,
*
1Department of Medicinal Chemistry
2Department of Anesthesiology
3Department of Biology
4Department of Pharmacology and Toxicology
University of Utah, Salt Lake City, UT 84112, USA
5Metabolomics Core Research Facility and Department of Biochemistry, University of Utah Health Sciences, Salt Lake City, UT 84112, USA
6Marine Science Institute
7Institute of Chemistry
University of the Philippines, Diliman, Quezon City 1101, Philippines
8Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health & Science University,
Beaverton, OR 97006, USA
*Correspondence: ews1@utah.edu
http://dx.doi.org/10.1016/j.chembiol.2012.10.019
SUMMARY
small molecules, although only a few systems have been studied
in any detail (Piel, 2009; Kubanek et al., 1997).
In the oceans, secondary metabolites often protect
otherwise poorly defended invertebrates, such as
shell-less mollusks, from predation. The origins of
these metabolites are largely unknown, but many of
them are thought to be made by symbiotic bacteria.
In contrast, mollusks with thick shells and toxic
venoms are thought to lack these secondary metab-
olites because of reduced defensive needs. Here, we
show that heavily defended cone snails also occa-
sionally contain abundant secondary metabolites,
g-pyrones known as nocapyrones, which are
Mollusks lacking shells are sometimes defended instead by
secondary metabolites (Benkendorff, 2010). Often, the mole-
cules seem to have a dietary origin (Fontana, 2006; Gavagnin
et al., 1994). However, a widely occurring group of mollusk
secondary metabolites, pyrones and structurally related polyke-
tides, are generated in the animals de novo, possibly even by the
mollusks themselves (Davies-Coleman and Garson, 1998; Marin
et al., 1999; Cimino et al., 1987; Ireland and Scheuer, 1979). The
close structural resemblance of these polyketides to bacterial
metabolites has led to the hypothesis that symbiotic bacteria,
and not the animals, produce the compounds in question
(Cutignano et al., 2009). This idea is complicated by the fact
synthesized by symbiotic bacteria. The bacteria, that there are many convergent biosynthetic routes to pyrones
(Busch and Hertweck, 2009), so the ultimate source is a matter
of debate. Molecular or genetic evidence is still lacking to defin-
Nocardiopsis alba CR167, are related to widespread
actinomycetes that we propose to be casual symbi-
itively tie production of any gastropod metabolite to symbiotic
bacteria or to any other source.
Mollusks with shells, such as cone snails and turrids (Terlau
onts of invertebrates on land and in the sea. The
natural roles of nocapyrones are unknown, but they
are active in neurological assays, revealing that
mollusks with external shells are an overlooked
source of secondary metabolite diversity.
´
and Olivera, 2004; Lopez-Vera et al., 2004), synthesize animal
genome-derived peptide toxins, both for predation and for
defense (Olivera, 2002). In contrast to their shell-less relatives,
mollusks with external shells are usually believed to be deficient
in secondary metabolites (Benkendorff, 2010), leading re-
searchers to avoid the animals as sources of new natural prod-
INTRODUCTION
Many marine animals are protected by arsenals of defensive ucts. For venom-containing mollusks, like cone snails, peptide
compounds, which have been exploited in the discovery of phar- toxins should provide ample defense against predators.
maceuticals (Putz and Proksch, 2010). These chemicals can be
Recently, we began to question whether cone snails might
divided into two groups: proteins and peptides (large molecules) also harbor symbiont-derived metabolites. If so, this might chal-
and secondary metabolites (small molecules). Protein-based lenge current ideas about distribution and roles of marine natural
toxins include venoms, which are synthesized by the animals products and suggest that otherwise protected animals are
themselves and are encoded in the animal genomes (Terlau good sources for discovery. Because by far most mollusks are
and Olivera, 2004). Small molecules are highly diverse in terms defended by shells, this would open a new area for compound
of their chemical structures, and they are more commonly asso- discovery. We reported that at least three species of cone snails
ciated with soft-bodied, ‘‘defenseless’’ animals, such as ascid- contain abundant associated actinomycete bacteria, which are
ians, sponges, and nudibranch mollusks (Blunt et al., 2011). In well known to produce secondary metabolites in culture (Peraud
contrast to the case with protein toxins, increasing evidence indi- et al., 2009). There are now many reports of actinomycetes iso-
cates that symbiotic bacteria synthesize at least some of these lated from marine animals. However, these actinomycetes have
Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved 73

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
Figure 1. Actinobacteria in C. rolani
(A) Live sample of C. rolani used in this study.
(B) Cultivated N. alba CR167 from C. rolani.
See also Tables S1 and S2.
not been tied to animal biology, and one hypothesis is that these
might usually be derived from very casually occurring bacteria or
even from spores. On land, actinomycetes have been shown to
form casual symbioses with a variety of insects, where they
secrete compounds implicated in defense against infection
(Schoenian et al., 2011; Kroiss et al., 2010). Because of the abun-
dance of actinomycetes in certain cone snails, we thought it
possible that actinomycetes might also play central roles in
marine organisms.
Here, we report a case in which actinomycete bacteria
synthesize polyketide pyrones in the cone snails, Conus rolani
and Conus tribblei. These results demonstrate that bacteria
produce gastropod compounds within their hosts, answering
a long-standing question about origins of mollusk metabolites.
The results also suggest that shelled mollusks are overlooked
sources of secondary metabolic diversity.
RESULTS
Figure 2. Structures of Nocapyrones H-Q (1–10) and A-C (11–13)
See also Figures S1–S3.
Neuroactive Nocapyrones from Cone-Snail-Associated
Bacteria
Strain N. alba CR167 was isolated from the venom duct of First, we examined the configuration of the C-6 OH, present in
C. rolani, collected in Mactan Island, Philippines, in 2007 with compounds 3, 6, and 8. The Mosher’s esters (Dale and Mosher,
informed consent and permission from local and national 1973) were synthesized and analyzed by 1H NMR, revealing that
governments, as previously reported (Figure 1) (Peraud et al., C-6 in all compounds was in the S configuration (Figure S2). We
2009). The extract from N. alba CR167 was neuroactive, as as- used the same method to examine the C-11 OH, present in
sayed by calcium imaging of dissociated dorsal root ganglion compounds 2, 6, and 11. In 2, C-11 was in the R configuration,
(DRG) neurons from mice. Activity was followed through a series whereas for 6 and 11, it was in the S configuration (Figure S2).
of chromatographic steps, leading to purification of 13 com-
We next sought to determine the configuration of the remote
pounds (Figure 2). We identified ten compounds, nocapyrones methyl groups at C-10. Compounds 6 and 11 were isolated as
H-Q (1–10), as well as the three previously described com- inseparable mixtures of diasteromers in the side chain. Careful
pounds, nocapyrones A-C (11–13) (Schneemann et al., 2010). examination showed that11wasalso previously isolated as adia-
Their structures were elucidated on the basis of one- and two- stereomeric mixture in the initial report of its structure (Schnee-
dimensional nuclear magnetic resonance (NMR) and by mass mann et al., 2010). The diastereomeric ratio of 6 was determined
spectrometry (Figure S1 available online; Table S1). Briefly, as 1.3:1 by integration of O-methine protons at d 3.62 and 3.55
high-resolution mass spectrometry was consistent with the (Figure S1), with a similar ratio observed for 11. Comparison of
molecular formulae of each molecule, whereas tandem mass the chemical shifts and coupling constants of O-methine protons
spectrometry (MS/MS) showed that the nocapyrones followed in 6 and 11 with literature values for (2R*,3S*)- and (2R*,3R*)-3-
a very consistent fragmentation pattern (Table S2) for g-pyrones methyltridecan-2-ol (Larsson et al., 2004) showed that the major
(Porter and Baldas, 1971). 13C and 1H resonances were assigned isomer in both 6 and 11 had a 10R*,11S* relative configuration.
on the basis of 1H, 13C, COSY, HSQC, and HMBC spectra (Fig- Initially, upon isolation we thought that this was likely due to
ure S1; Table S1). partial racemization at the OH-bearing C-11 position, but the
Because configurational information is lacking for this Mosher’s analysis described above revealed that only single
compound class, we determined the stereochemistry of 1-13. C-11 isomers were present (Figure S2). Thus, compounds 6a
74 Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
Table 1. Nocapyrones Detected in Methanolic Extracts of Conus rolani
Pyrones Detected
1
3
12
Mean SD
(mg/g)a
Mean SD
Mean SD
Tissue
Date Collected
Sept. 2009
Sept. 2010
Nov. 2011
July 2011
d (ppm)b
1.67
(mg/g)
d (ppm)
À0.87
À0.34
À0.68
2.05
(mg/g)
d (ppm)
1.22
1
Whole snail (n = 1)c
Whole snail (n = 3)c
Whole snail (n = 4)c
Venom duct (n = 20)c
12.4 2.7
7.84 0.34
nd
1,250 154
507 31
129 37
11.0 2.2d
N.D.
1.57 0.26
10.2 1.5
nd
2
0.97
0.87
3
4
0.404 0.012d
1.30
0.86
0.366 0.095d
1.88
1.44
4a
Other anatomical
regions (n = 2)
Mucus (n = 4)c
July 2011
nd
nd
5
6
Oct. 2011
Mar. 2012
44.7 5.2e
nd
105 7.2e
nd
À1.77
31.1 2.48e
nd
Whole snail and
mucus (n = 20)c
Mean over three independent measurements. Data analysis was done using Analyst QS software 2.0. nd, below detection limit. Statistical limit of
quantification: 0.29 ng/ml; practical detection limit: 0.10 ng/ml. See also Figures S4 and S5.
aMicrogram compound per gram of snail tissue dry weight (each snail weighs ꢀ1 g).
bParts per million (ppm) error of high-resolution mass spectrometric measurement.
cn, number of samples averaged to determine quantity but with each individual sample measured separately. In these cases, each snail contained the
compounds, and averaging was done for ease of presentation.
dNumbers reflect microgram per venom duct rather than per gram dry weight.
eNumbers reflect microgram per snail rather than per gram dry weight.
and 11a have the 10R, 11S configuration, whereas 6b and 11b had demonstrated that actinobacteria live in the foot of snails,
have the 10S, 11S configuration.
indicating that cultivated strains, such as Nocardiopsis sp., might
Using Mosher’s method, compound 7 was shown to be be alive within the snails, rather than existing as inactive spores
present as an enatiomeric mixture, with a S:R ratio of 10:1 (Fig- (Peraud et al., 2009). However, this hypothesis still seemed
ure S3). Although in this case a primary alcohol was esterified, highly speculative because polyketides had never before been
whereas the Mosher’s method is used mainly for secondary reported from thickly shelled mollusks, such as cone snails.
alcohols, an excellent literature precedent was available for Therefore, finding the Nocardiopsis pyrones within whole snail
compounds with related functional groups: (2S)-2-methylhexyl tissue would provide strong support for the hypothesis.
(2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate and (2R)-2-
To test this hypothesis, we extracted the whole animals with
methylhexyl (2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate ethanol and analyzed their organic content. Surprisingly, 1, 3,
(Guintchin and Bienz, 2003). The Mosher’s ester of the major and 12 were among the major organic components that could
isomer 7a exhibited chemical shifts of 4.24, 4.08 (AB of ABX, be isolated from an individual snail collected in 2009. This
JAB = 10.7 Hz, JAX = 6.6 Hz, JBX = 5.7 Hz, OCH2), which were prompted us to perform a more detailed chemical analysis (Fig-
very similar to those reported in the literature precedent for the ure S4), collecting and analyzing snails from the same site near
S isomer (Figure S3). Likewise, 7b exhibited chemical shifts Sogod, Cebu Island, during the period of 2009–2012 and deter-
consistent with the R isomer. Thus, the major 7a was present mining the tissue localization and relative abundance of com-
as the S isomer.
pounds (Table 1). We synthesized a 13C-labeled pyrone as an
We wished to determine the remote methyl center of 5 directly, internal control to accurately calibrate the quantity of com-
but no functional handle or standard compound was available. A pounds and performed control experiments to accurately deter-
closely related fatty acid in the S configuration has a reported mine concentrations in the snails (Figure S5). To ensure that
optical rotation of [a]D = + 8.0 (Sonnet et al., 1990), whereas 5 results would not be compromised by laboratory contamination,
has a measured rotation of [a]D = + 9.0. In addition, the major work was divided between two geographical locations.
isomers of related compounds 6 and 11 are in the S configura-
Compounds 1, 3, and 12 were variable in abundance in
tion. Thus, we tentatively propose that 5 is likely also in the S C. rolani and C. tribblei samples and were sometimes not
configuration.
present (Table 1; Figure S5). When present, the compounds
were relatively abundant, totaling between 0.01%–0.1% of snail
dry weight (not including the shell). Linearity was demonstrated
Nocapyrones from Methanolic Extracts of C. rolani
We noticed that nocapyrones were structurally related to previ- from calibration curves in the range 0.010 to 10.0 mg/ml with
ously reported polyketides from other gastropod mollusks. correlation coefficients of at least 0.9996. Percent recovery of in-
Several pyrones are known to be made within mollusks based jected standard (1 mg) from live snails was found to be 87.78%
upon labeling studies (Ireland and Scheuer, 1979; Di Marzo 3.71% (n = 3). This abundance is on par for what is commonly
et al., 1991), but whether by the animal, symbiotic bacteria, or found for other marine animal metabolites in better-studied
other organism was unknown. We hypothesized that N. alba systems, such as sponges, ascidians, and soft-bodied mollusks,
CR167 synthesizes pyrones within cone snail tissues. We already indicating that the compounds are present in a physiologically
Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved 75

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
relevant concentration range. Variability of secondary metabo- 2007). An authentic substrate was generated by chemical deme-
lites between samples is frequently encountered in marine thylation of the natural products. Close structural homologs
natural products (Donia et al., 2011).
were also available for assay. Although the natural substrates
We investigated the tissue specificity of nocapyrones. Com- were efficiently methylated with the anticipated regiochemistry,
pounds were only found in two locations and were absent else- chemically similar compounds (4-hydroxy-3,6-dimethyl-2-
where (Table 1). Nocapyrones appeared to be largely localized pyrone, 4-hydroxy-6-methyl-2-pyrone, and germicidin A) were
to the mucus, with a small amount (ꢀ1% of the compounds) not substrates (Figure 4). The narrow substrate selectivity of
found in the venom duct. Pyrones and other polyketides have this enzyme strongly supports the assignment of ncp as the
previously been isolated from the mucus of soft-bodied mollusks, nocapyrones biosynthesis cluster. Genetic knockout data would
where they are proposed to function in defense or communica- be desirable to reinforce this finding, but in initial experiments the
tion (Di Marzo et al., 1991; Sleeper and Fenical, 1977).
strain was resistant to transformation by standard methods
(Kieser et al., 2000).
On this basis, we proposed that ncp (deposited in GenBank,
Origin of Nocapyrones in Cone Snail
The data described above strongly supported the hypothesis JN792621) was responsible for pyrone synthesis (Figure 3).
that bacteria produce cone snail pyrones, but there are many ncp encodes a cluster of four genes, ncpA (oxidoreductase),
potential caveats that necessitated further study (Schmidt, ncpB (methyltransferase), ncpC (PKS), and ncpD (putative free-
2008). For example, convergent evolution or horizontal gene standing acyltransferase). The correct assembly of this gene
transfer could lead to more complex scenarios (Schmidt, cluster was confirmed by PCR, as well as by a nearly identical
2008). To tie the bacteria to pyrone production within the and syntenic cluster encoded in the chromosome of the recently
animals, we identified the probable pyrone biosynthetic gene deposited N. alba ATCC BAA-2165. Based upon the domain
cluster and showed that both this cluster and N. alba bacteria architecture of NcpC, and in analogy to other pyrone biosyn-
were present in the snails themselves.
thetic pathways, a hypothetical biogenetic scheme can be
At least three convergent routes lead to the biosynthesis of py- proposed (Figure 3). First, diverse fatty acyl CoA esters are
rones in nature (Busch and Hertweck, 2009). Therefore, to iden- loaded onto NcpC. Subsequently, the PKS domains act itera-
tify the pyrone biosynthetic gene cluster, we sequenced the tively, extending the fatty acid with two units of methylmalonyl
genome of N. alba CR167 to an average of 250 3 coverage. CoA. As is found with some other PKS proteins, in the absence
The resulting genome was assembled into 353 contigs (N50 = of a thioesterase cleavage of a diketoester proceeds in tandem
44.1 kbp); because of this fragmentation we analyzed both with pyrone formation (Frank et al., 2007). Finally, the resulting
assembled contigs and raw reads to find the pyrones gene products are substrates for O-methylation by NcpB. NcpA might
cluster. The central metabolic genes in the strain were similar to catalyze C-oxidation. Existing data do not strongly define the
those from the previously sequenced Nocardiopsis dassonvillei role for NcpD, although it may be involved in starter unit loading
(GenBank CP002040) (Sun et al., 2010), although both synteny or product offloading. It should be noted that other biosynthetic
and sequence identity were relatively low between the genomes. routes are possible. We favor this scheme because the NcpC
Very recently, the genome sequence of Nocardiopsis alba ATCC acyltransferase is likely methylmalonate specific, and the side
BAA-2165, cultivated from the digestive tract of honeybees in chain variation in the pyrone series is strongly reminiscent of
Ohio, was published in GenBank (CP003788). This genome the normal mixture found in actinomycete fatty acids. Finally,
was >95% DNA sequence identical to that of N. alba CR167 a subtle point is that only the linear side-chain pyrones were iso-
and shared 16 out of 18 identified biosynthetic gene clusters, lated from cone snails, which is expected in animals if the source
usually at >99% DNA sequence identity.
Pyrones are known to be made by various types of polyketide
fatty acids originate from primary metabolism.
With the biosynthetic cluster in hand, PCR primers (Figure S7)
synthase (PKS) proteins. Using BLAST and antiSMASH (Me- were designed to specifically detect the presence of the same
dema et al., 2011) analysis, only three PKS gene clusters were genes in whole snail tissues. Indeed, both ncpB and ncpC could
present in N. alba CR167, including a type II PKS and two type be amplified from snail tissue, as could the N. alba CR167 16S
I PKS pathways. No type III PKSs were present. Nearly identical rRNA gene sequence (Figure 5). The variable nature of
pathways were also found in the well-assembled N. alba ATCC compound production and the relative difficulty of obtaining
BAA-2165 genome. Of the three PKS clusters, only one con- samples (the snails live at À70 m) precluded more detailed
tained a methyltransferase. That PKS cluster was also the only colocalization studies. Indeed, the characteristics of N. alba
one that appeared to have the correct domain specificity and CR167 are consistent with a lifestyle that is not host restricted.
module architecture to produce pyrones. In addition, there is N. alba CR167 was readily cultured and maintained in the labo-
a known pyrone g-O-methyltransferase from the jerangolid ratory. Therefore, these bacteria are casual associates of cone
gene cluster (Julien et al., 2006). BLAST searching for homologs snails, rather than obligate symbionts. Finally, previously we
in the genome revealed only a single significant hit, also corre- showed that actinobacteria specifically inhabit mucus-gener-
sponding to this PKS-clustered methyltransferase. Thus, ating cells within C. rolani (Peraud et al., 2009). This location is
genomic data strongly implicated the identified ncp cluster as consistent with the chemical results found here.
being responsible for nocapyrone biosynthesis (Figure 3).
To provide initial chemical evidence in support of the bio- Cone Snails as a Source of Small Molecules for Bioactive
informatics results, the jerangolid-like methyltransferase NcpB Compound Discovery
was produced in Escherichia coli, purified (Figure S6), and Nocapyrones modulated nerve cell depolarization, with the most
used in enzyme assays with various substrates (Nelson et al., active compounds achieving an IC50 of 2 mM. Nocapyrones
76 Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
Figure 3. Organization of the Nocapyrone Biosynthetic Gene Clusters and Model for Nocapyrone Biosynthesis
KS, b-keto acyl synthase; AT, acyl transferase; ACP, acyl carrier protein; mMCoA, methyl malonyl CoA; MT, methyltransferase; Ox, oxidoreductase. This scheme
shows the hypothetical biogenesis (see text). See also Figure S6.
B (12) and H (1) were active against nearly all DRG neuronal cell or a panel of ꢀ60 human channels and receptors (http://pdsp.
types at 50 mM in the calcium-imaging assay, whereas 3 was med.unc.edu/). We also tested compounds 1, 5, and 12 against
inactive in the assay. We use the DRG assay because it provides a panel of human cells overexpressing various transient receptor
a rapid means to test response of diverse receptors in diverse potential (TRP) channels, and they were able to activate or inhibit
neuronal subtypes (Teichert et al., 2012a, 2012b; Lin et al., Ca2+ flux into those cells with IC50s between 2 and 70 mM, de-
2011). DRG neurons are known to be heterogeneous, with pending upon the agent and channel subtype (Table S3).
a variety of different cell types that respond to different stimuli. Notably, the menthol-sensitive TRPM8 channel was inhibited,
Using the calcium-imaging assay, cell types can be identified whereas TRPA1 was activated. This is consistent with findings
by their differential responses to discrete reagents (Teichert using the DRG assay, where menthol-sensitive cells were
et al., 2012a).
inhibited, whereas TRPA1 is abundantly expressed in capsa-
Depending on the DRG neuronal cell type, nocapyrones either icin-sensitive neurons. However, the broad effects of the noca-
inhibited or amplified responses that were elicited by depolariz- pyrones on TRP channels suggests that they may affect an
ing the cells with a brief application of high extracellular potas- underlying channel-regulating element that leads to cell-type
sium (KCl pulse). For example, both compounds 1 and 12 specific effects, rather than acting directly on the channels or
partially blocked depolarization-elicited (i.e., KCl-elicited) cell-surface receptors tested.
increases in cytoplasmic calcium in large-diameter cells that
Compounds 1 and 12 were also tested against the human
were capsaicin resistant, whereas they amplified the KCl-elicited breast adenocarcinoma (MCF-7) and Chinese hamster ovary
increases in cytoplasmic calcium of small-diameter, capsaicin- (AA8) cell lines. Against MCF-7, 1 and 12 were cytotoxic with
sensitive cells (Figure 6). In additional tests, the effects of IC50 values of 8.7 and 22.2 mM, respectively, whereas 3 was inac-
compound 1 did not appear to correlate with acetylcholine- tive against MCF-7 (>100 mM), and against AA8 only 1 was cyto-
sensitivity, whereas an amplification was observed consistently toxic with an IC50 of 10.2 mM. By contrast, the compounds were
in small, capsaicin- and histamine-sensitive cells. A near- not bacteriostatic or bactericidal. Schneemann et al. (2010) previ-
complete block was observed in a subset of menthol-sensitive ously showed that nocapyrones A-D were inactive against an
cells (Figure S8). The compounds did not affect sodium channels even broader panel of bacteria and the yeast Candida glabrata.
Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved 77

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
Figure 4. NcpB Is the Nocapyrone Ο-methyltransferase
High-performance liquid chromatography (HPLC) traces are shown with detection at 228 nm.
(A) Standards of pure 9 and 9a.
(B) Enzyme assay containing 9a, NcpB, and SAM.
(C) Control enzyme assay lacking SAM.
(D) Control enzyme assay with boiled NcpB.
(E–G) Other pyrone substrates were not accepted by the enzyme, using the same reaction conditions as shown in (B).
DISCUSSION
eukaryotes a-methylated polyketides usually arise from later
methylation of polyketide intermediates.
Molecular genetics can provide the definitive answer to the
Here, we show that bacteria produce pyrone polyketides in cone
snails. Pyrones and related polyketides have been isolated from source question for marine natural products (Piel, 2009). Here,
many different mollusks from many habitats, including diverse we used multiple types of chemical and molecular biological
sacoglossan, siphonarid, and cephalispidean mollusks (Gavag- evidence to show that N. alba CR167 bacteria make in the
nin et al., 1994; Davies-Coleman and Garson, 1998; Ireland cone snail species, C. rolani and C. tribblei. These multiple
and Scheuer, 1979; Di Marzo et al., 1991, 1993; Sleeper and methods were required to demonstrate that the bacteria are
Fenical, 1977; Manker et al., 1988; Darias et al., 2006), where important to their hosts. This is not a given; indeed, bacteria culti-
they are produced de novo (Ireland and Scheuer, 1979; Sleeper vated from animals are most likely to be irrelevant to the core
and Fenical, 1977; Manker et al., 1988). However, this informa- biology of the animals (Donia et al., 2011; Klepzig et al., 2009).
tion does not prove the source. For example, sacoglossans eat As an example, actinomycetes have been previously isolated
eukaryotic green algae and employ both the chloroplasts and from many different marine animals, where they have been
algal mRNA to survive on sunlight for extended periods (Rumpho shown to be an excellent source of promising drug leads (Blunt
et al., 2000; Mujer et al., 1996). Feeding studies demonstrate that et al., 2011). However, in no prior case has an actinomycete
sacoglossan pyrones are labeled by carbonate so that they obvi- been directly tied to actual function in marine animals, to the
ously arise from intermediates of photosynthesis mediated by best of our knowledge. Prior knowledge (Donia et al., 2011)
the hosted chloroplasts (Ireland and Scheuer, 1979). The pyrone suggests that the majority of such actinomycete isolates may
C-methyl groups arise from propionate, which might indicate not actively grow with their hosts and so do not contribute abun-
a bacterial source (Di Marzo et al., 1991; Cutignano et al., dant chemicals to the whole animals. Tying individual cultivated
2012). This is due to the fact that a-methylated polyketides often bacterial strains to function in vivo requires careful study.
arise from incorporation of the polyketide precursor methylmal- Actinomycete symbionts are well known to produce
onate (propionate) in bacteria, whereas in animals and other secondary metabolites used by terrestrial insects (Barke et al.,
78 Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
Figure 5. Genes for Nocapyrone Synthesis Are Found in Cone Snail
Tissue
The N. alba CR167 ncpB, ncpC, and 16S rRNA genes were amplified from both
C. rolani and C. tribblei metagenome DNA by nested PCR experiments. The
PCR products were confirmed to be identical to the positive controls by
Sanger sequencing the gel-extracted PCR products.
(A) Primers noc16s_Fwd and noc16s_Rev.
(B) Primers MT_in_Fwd and MT_in_Rev.
(C) Primers PKS_in_Fwd and PKS_in_Rev. Template DNA: (1) C. tribblei; (2)
C. rolani; (3) N. alba CR167; (4) no DNA control; 0) DNA ladder.
See also Figure S7.
2010), whereas in most marine symbioses so far studied
secondary metabolites are produced by proteobacteria or cya-
nobacteria. In ants, the actinomycete symbionts and their
produced chemicals vary over time and space, even in single
types of animals (Barke et al., 2010). Whether the symbionts
are variable or stable, the resulting compounds play important
ecological roles (Currie et al., 2003; Seipke et al., 2011). The
symbiosis reported here has many similarities to the casual
actinomycete symbioses reported on land but extends these
observations to the marine environment. It is noteworthy that
our data were acquired from repeated sampling of wild popula-
tions so that the results are relevant to the chemical ecology of
the organisms.
In 2010, Patil and colleagues isolated N. alba ATCC BAA-2165
from the digestive tract of honeybees in Ohio (GenBank:
CP003788.1) (Patil et al., 2010). The bacteria could be reliably
cultivated as one of the major putative symbionts over all
seasons in a year. The strain showed modest antibiotic activity,
although for unknown reasons. While this manuscript was under
review, the genome of this strain was released, showing that
overall it is nearly identical to that of N. alba CR167. This
extremely close relationship shows that N. alba is a common
but casual symbiont of both marine and terrestrial invertebrates.
Moreover, the more distantly related N. dassonvillei is also an
animal associate, including very rarely acting as a human
pathogen (Penn and Jensen, 2012; Lejbkowicz et al., 2005).
Finally, nocapyrones 11–13 were previously reported from a
Nocardiopsis strain isolated from sponges. We propose that
N. alba, and possibly related Nocardiopsis strains, are widely
occurring, casual symbionts of diverse invertebrates and that
pyrones play a role in these symbioses. As the sponge and
honeybee cases have not been functionally studied, it is
unknown whether pyrones are produced in other invertebrates
or what the roles of these symbionts might be.
Figure 6. Activity of Compound 12 Observed by Calcium Imaging of
Dissociated DRG Neurons in Culture
Each trace is the response of a single neuron. Responses from ꢀ100 neurons
were monitored individually and simultaneously in a given experimental trial.
Selected traces are shown from a single experimental trial. The y axis is
a measure of relative intracellular (cytoplasmic) calcium concentration, [Ca2+]i,
obtained by standard ratiometric calcium-imaging techniques (i.e., ratio of
340 nm/380 nm excitation while monitoring fluorescence emission at 510 nm).
The x axis is time in minutes. Increases in [Ca2+]i were elicited by briefly de-
polarizing the neurons with 25 mM potassium (KCl pulse) at regular 7 min
intervals, as indicated by vertical arrows. Each KCl pulse elicited an increase in
[Ca2+
] (by activating voltage-gated calcium channels) that is observed as
i
a peak in each trace. Capsaicin (300 nM) was applied to the neurons for 1 min
at the end of the experiment, as indicated by the black circle. After the fourth
KCl pulse, the compound was applied to the cells for 6 min (horizontal bar). In
many small-diameter, capsaicin-sensitive DRG neurons, the compound
caused an amplification of the calcium-transient elicited by a KCl pulse
(bottom three traces) or directly elicited a calcium-transient (fourth trace from
the bottom). In contrast, in many large-diameter, capsaicin-resistant neurons,
the compound partially blocked the calcium-transient elicited by a KCl pulse
(top four traces). All effects of the compound were reversible, as shown.
See also Figure S8 and Table S3.
Our results show that mollusks that are heavily defended both
with a heavy shell and toxic venom can also harbor bioactive
small molecules in their mucus. These compounds were present
at concentrations relevant to chemical defense and that are the snails live in relatively deep water (À70 m) and do not survive
below those needed to directly affect mammalian cells and well in aquaria. However, related molecules from shell-less
neurons. Our system is challenging for ecology studies in that mollusks have been experimentally determined to serve roles
Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved 79

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
data transfer and Wes Tolman (University of Utah), Joshua Orvis, Kevin Ga-
lens, and Chris Hemmerich (all of the The Joint Genome Institute) for helping
us to install the Ergatis software and bioinformatics database in-house.
such as defense, regeneration, and communication (Gavagnin
et al., 1994; Davies-Coleman and Garson, 1998; Ireland and
Scheuer, 1979; Di Marzo et al., 1991, 1993; Sleeper and Fenical,
1977; Manker et al., 1988; Darias et al., 2006). Further study is
required to determine whether the pyrones play these or other
roles within cone snail mucus. It should be clear that, in this con-
text, communication has many possible meanings. For example,
the compounds speculatively could play a role in communication
between snails, between snails and bacteria, or even between
bacteria in roles such as quorum sensing.
Received: July 18, 2012
Revised: October 11, 2012
Accepted: October 24, 2012
Published: January 24, 2013
REFERENCES
Barke, J., Seipke, R.F., Gru¨ schow, S., Heavens, D., Drou, N., Bibb, M.J., Goss,
R.J.M., Yu, D.W., and Hutchings, M.I. (2010). A mixed community of actinomy-
cetes produce multiple antibiotics for the fungus farming ant Acromyrmex
octospinosus. BMC Biol. 8, 109.
SIGNIFICANCE
Otherwise defenseless, mollusks are known to be good
sources of small molecules for drug discovery. We show
that even thickly shelled gastropods are a good source of
secondary metabolites with medicinal potential. Dogma
suggested that these mollusks may be poor sources of
secondary metabolites, but mollusks with shells have not
been widely explored recently. Given the improvement in
analytical methods since the dawn of marine natural prod-
ucts research, gastropods with shells warrant a second
look as a source of new compounds.
Benkendorff, K. (2010). Molluscan biological and chemical diversity:
secondary metabolites and medicinal resources produced by marine
molluscs. Biol. Rev. Camb. Philos. Soc. 85, 757–775.
Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., and Prinsep, M.R.
(2011). Marine natural products. Nat. Prod. Rep. 28, 196–268.
Busch, B., and Hertweck, C. (2009). Evolution of metabolic diversity in polyke-
tide-derived pyrones: using the non-colinear aureothin assembly line as
a model system. Phytochemistry 70, 1833–1840.
Cimino, G., Sodano, G., and Spinella, A. (1987). New propionate-derived
metabolites from Aglaja depicta and from its prey Bulla striata (opisthobranch
mollusks). J. Org. Chem. 52, 5326–5331.
Bacteria are often thought to be the ultimate source of
small molecules in animals, but this has only been closely
investigated in a relatively small number of studies. Although
many bacteria have been cultivated from marine animals, it
is hard to determine whether those bacteria play a role in
the biology of the animals and whether they produce
compounds within their hosts. Here, we provide evidence
that actinomycetes make secondary metabolites in marine
animals, using a series of methods to rigorously tie bacterial
biosynthesis to phenotype within animals. These relation-
ships are, surprisingly, closely related to interactions found
in very distant habitats on land, showing a widespread pene-
tration of casual actinomycete symbionts in invertebrates.
Finally, pyrones and related polyketides are a widespread
family of mollusk compounds, for which the ultimate biosyn-
thetic source was not known. Here, we show that these
compounds are produced by symbiotic bacteria within
cone snails. Although further study is required to determine
whether other mollusk polyketides are bacterially synthe-
sized, the methods we use are widely applicable.
Cutignano, A., Cimino, G., Villani, G., and Fontana, A. (2009). Shaping the
polypropionate biosynthesis in the solar-powered mollusc Elysia viridis.
ChemBioChem 10, 315–322.
Cutignano, A., Villani, G., and Fontana, A. (2012). One metabolite, two path-
ways: convergence of polypropionate biosynthesis in fungi and marine
molluscs. Org. Lett. 14, 992–995.
Currie, C.R., Wong, B., Stuart, A.E., Schultz, T.R., Rehner, S.A., Mueller, U.G.,
Sung, G.H., Spatafora, J.W., and Straus, N.A. (2003). Ancient tripartite coevo-
lution in the attine ant-microbe symbiosis. Science 299, 386–388.
Dale, J.A., and Mosher, H.S. (1973). Nuclear magnetic resonance enantiomer
regents. Configurational correlations via nuclear magnetic resonance
chemical shifts of diastereomeric mandelate, O-methylmandelate, and
a-methoxy-a-trifluoromethylphenylacetate (MTPA) esters. J. Am. Chem.
Soc. 95, 512–519.
Darias, J., Cueto, M., and Dı´az-Marrero, A.R. (2006). The chemistry of marine
pulmonate gastropods. Prog. Mol. Subcell. Biol. 43, 105–131.
Davies-Coleman, M.T., and Garson, M.J. (1998). Marine polypropionates. Nat.
Prod. Rep. 15, 477–493.
Di Marzo, V., Vardaro, R.R., De Petrocellis, L., Villani, G., Minei, R., and Cimino,
G. (1991). Cyercenes, novel pyrones from the ascoglossan mollusk Cyerce
cristallina. Tissue distribution, biosynthesis and possible involvement in
defense and regenerative processes. Cell. Mol. Life Sci. 47, 1221–1227.
EXPERIMENTAL PROCEDURES
Di Marzo, V., Marin, A., Vardaro, R.R., De Petrocellis, L., Villani, G., and Cimino,
G. (1993). Histological and biochemical bases of defense mechanisms in four
species of Polybranchioidea ascoglossan mollusks. Mar. Biol. 117, 367–380.
See the Supplemental Experimental Procedures.
Donia, M.S., Fricke, W.F., Ravel, J., and Schmidt, E.W. (2011). Variation in
tropical reef symbiont metagenomes defined by secondary metabolism.
PLoS ONE 6, e17897.
SUPPLEMENTAL INFORMATION
Supplemental Information includes eight figures, three tables, and Supple-
mental Experimental Procedures and can be found with this article online at
http://dx.doi.org/10.1016/j.chembiol.2012.10.019.
Fontana, A. (2006). Biogenetic proposals and biosynthetic studies on
secondary metabolites of opisthobranch molluscs. Prog. Mol. Subcell. Biol.
43, 303–332.
¨
¨
Frank, B., Wenzel, S.C., Bode, H.B., Scharfe, M., Blocker, H., and Muller, R.
(2007). From genetic diversity to metabolic unity: studies on the biosynthesis
of aurafurones and aurafuron-like structures in myxobacteria and streptomy-
cetes. J. Mol. Biol. 374, 24–38.
ACKNOWLEDGMENTS
This work was funded by an ICBG grant (U01TW008163) from Fogarty
(National Institutes of Health [NIH]). We thank the government of the
Philippines and the community of Mactan Island for permission to conduct
this study. We thank Brian Dalley and Brett Milash (University of Utah Geno-
mics Core Facility) for sequencing the genome of CR167 and aiding with
Gavagnin, M., Marin, A., Mollo, E., Crispino, A., Villani, G., and Cimino, G.
(1994). Secondary metabolites from the Mediterranean Elysioidea: origin and
biological role. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 108, 107–115.
80 Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
A Bacterial Source for Mollusk Pyrone Polyketides
Guintchin, B., and Bienz, S. (2003). Stereoconvergent preparation of chiral
vinylsilanes by cuprate substitution of a-acetoxyallylsilanes. Application to
the synthesis of (S)-(+)-bishomomanicone. Tetrahedron 59, 7527–7533.
Nelson, J.T., Lee, J., Sims, J.W., and Schmidt, E.W. (2007). Characterization of
SafC, a catechol 4-O-methyltransferase involved in saframycin biosynthesis.
Appl. Environ. Microbiol. 73, 3575–3580.
Olivera, B.M. (2002). Conus venom peptides: reflections from the biology of
Ireland, C., and Scheuer, P.J. (1979). Photosynthetic marine mollusks: in vivo
14c incorporation into metabolites of the sacoglossan Placobranchus ocella-
tus. Science 205, 922–923.
clades and species. Annu. Rev. Ecol. Syst. 33, 25–47.
Patil, P.B., Zeng, Y., Coursey, T., Houston, P., Miller, I., and Chen, S. (2010).
Isolation and characterization of a Nocardiopsis sp. from honeybee guts.
FEMS Microbiol. Lett. 312, 110–118.
Julien, B., Tian, Z.Q., Reid, R., and Reeves, C.D. (2006). Analysis of the ambru-
ticin and jerangolid gene clusters of Sorangium cellulosum reveals unusual
mechanisms of polyketide biosynthesis. Chem. Biol. 13, 1277–1286.
Penn, K., and Jensen, P.R. (2012). Comparative genomics reveals evidence of
marine adaptation in Salinispora species. BMC Genomics 13, 86.
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A. (2000).
Practical Streptomyces Genetics (Norwich: The John Innes Foundation).
Peraud, O., Biggs, J.S., Hughen, R.W., Light, A.R., Concepcion, G.P., Olivera,
B.M., and Schmidt, E.W. (2009). Microhabitats within venomous cone snails
contain diverse actinobacteria. Appl. Environ. Microbiol. 75, 6820–6826.
Klepzig, K.D., Adams, A.S., Handelsman, J., and Raffa, K.F. (2009).
Symbioses: a key driver of insect physiological processes, ecological interac-
tions, evolutionary diversification, and impacts on humans. Environ. Entomol.
38, 67–77.
Putz, A., and Proksch, P. (2010). Chemical defence in marine ecosystems.
Annual Plant Rev. 39, 162–213.
Piel, J. (2009). Metabolites from symbiotic bacteria. Nat. Prod. Rep. 26,
Kroiss, J., Kaltenpoth, M., Schneider, B., Schwinger, M.G., Hertweck, C.,
Maddula, R.K., Strohm, E., and Svatos, A. (2010). Symbiotic Streptomycetes
provide antibiotic combination prophylaxis for wasp offspring. Nat. Chem.
Biol. 6, 261–263.
338–362.
Porter, Q.N., and Baldas, J. (1971). Mass Spectrometry of Heterocyclic
Compounds (New York: Wiley-Interscience), 139–147.
Rumpho, M.E., Summer, E.J., and Manhart, J.R. (2000). Solar-powered sea
Kubanek, J., Graziani, E.I., and Andersen, R.J. (1997). Investigations of terpe-
noid biosynthesis by the dorid nudibranch Cadlina luteomarginata. J. Org.
Chem. 62, 7239–7246.
slugs. Mollusc/algal chloroplast symbiosis. Plant Physiol. 123, 29–38.
Schneemann, I., Ohlendorf, B., Zinecker, H., Nagel, K., Wiese, J., and Imhoff,
J.F. (2010). Nocapyrones A-D, g-pyrones from a Nocardiopsis strain isolated
from the marine sponge Halichondria panicea. J. Nat. Prod. 73, 1444–1447.
Larsson, M., Galandrin, E., and Ho¨ gberg, H.-E. (2004). Diastereoselective
addition of organozinc reagents to 2-alkyl-3-(arylsulfanyl)propanals.
Tetrahedron 60, 10659–10669.
Schmidt, E.W. (2008). Trading molecules and tracking targets in symbiotic
interactions. Nat. Chem. Biol. 4, 466–473.
Lejbkowicz, F., Kudinsky, R., Samet, L., Belavsky, L., Barzilai, M., and
Predescu, S. (2005). Identification of Nocardiopsis dassonvillei in a blood
sample from a child. Am. J. Infect. Dis. 1, 1–4.
Schoenian, I., Spiteller, M., Ghaste, M., Wirth, R., Herz, H., and Spiteller, D.
(2011). Chemical basis of the synergism and antagonism in microbial
communities in the nests of leaf-cutting ants. Proc. Natl. Acad. Sci. USA
108, 1955–1960.
Lin, Z., Reilly, C.A., Antemano, R., Hughen, R.W., Marett, L., Concepcion, G.P.,
Haygood, M.G., Olivera, B.M., Light, A., and Schmidt, E.W. (2011).
Nobilamides A-H, long-acting transient receptor potential vanilloid-1
(TRPV1) antagonists from mollusk-associated bacteria. J. Med. Chem. 54,
3746–3755.
Seipke, R.F., Barke, J., Brearley, C., Hill, L., Yu, D.W., Goss, R.J.M., and
Hutchings, M.I. (2011). A single Streptomyces symbiont makes multiple
antifungals to support the fungus farming ant Acromyrmex octospinosus.
PLoS ONE 6, e22028.
Lo´ pez-Vera, E., Heimer de la Cotera, E.P., Maillo, M., Riesgo-Escovar, J.R.,
Olivera, B.M., and Aguilar, M.B. (2004). A novel structural class of toxins: the
methionine-rich peptides from the venoms of turrid marine snails (Mollusca,
Conoidea). Toxicon 43, 365–374.
Sleeper, H.L., and Fenical, W. (1977). Navenones A-C: trail-breaking alarm
pheromones from the marine opisthobranch Navanax inermis. J. Am. Chem.
Soc. 99, 2367–2368.
Sonnet, P.E., Gazzillo, J.A., Dudley, R.L., and Boswell, R.T. (1990). Synthesis
and characterization of enantiomers of 5- and 6-methyloctanoic acids. Chem.
Phys. Lipids 54, 205–214.
Marin, A., Alvarez, L.A., Cimino, G., and Spinella, A. (1999). Chemical defence
in cephalaspidean gastropods: origin, anatomical location and ecological role.
J. Molluscan Stud. 65, 121–131.
Sun, H., Lapidus, A., Nolan, M., Lucas, S., Del Rio, T.G., Tice, H., Cheng, J.F.,
Tapia, R., Han, C., Goodwin, L., et al. (2010). Complete genome sequence of
Nocardiopsis dassonvillei type strain (IMRU 509). Stand. Genomic Sci. 3,
325–336.
Manker, D.C., Garson, M.J., and Faulkner, D.J. (1988). De novo biosynthesis of
polypropionate metabolites in the marine pulmonate Siphonaria denticulata.
J. Chem. Soc. Chem. Commun. 16, 1061–1062.
Teichert, R.W., Smith, N.J., Raghuraman, S., Yoshikami, D., Light, A.R., and
Olivera, B.M. (2012a). Functional profiling of neurons through cellular neuro-
pharmacology. Proc. Natl. Acad. Sci. USA 109, 1388–1395.
Medema, M.H., Blin, K., Cimermancic, P., de Jager, V., Zakrzewski, P.,
Fischbach, M.A., Weber, T., Takano, E., and Breitling, R. (2011). antiSMASH:
rapid identification, annotation and analysis of secondary metabolite biosyn-
thesis gene clusters in bacterial and fungal genome sequences. Nucleic
Acids Res. 39 (Web Server issue), W339–W346.
Teichert, R.W., Raghuraman, S., Memon, T., Cox, J.L., Foulkes, T., Rivier, J.E.,
and Olivera, B.M. (2012b). Characterization of two neuronal subclasses
through constellation pharmacology. Proc. Natl. Acad. Sci. USA 109,
12758–12763.
Mujer, C.V., Andrews, D.L., Manhart, J.R., Pierce, S.K., and Rumpho, M.E.
(1996). Chloroplast genes are expressed during intracellular symbiotic associ-
ation of Vaucheria litorea plastids with the sea slug Elysia chlorotica. Proc. Natl.
Acad. Sci. USA 93, 12333–12338.
Terlau, H., and Olivera, B.M. (2004). Conus venoms: a rich source of novel ion
channel-targeted peptides. Physiol. Rev. 84, 41–68.
Chemistry & Biology 20, 73–81, January 24, 2013 ª2013 Elsevier Ltd All rights reserved 81