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Metagenomic natural product discovery in PNAS PLUS provides evidence for a of biosynthetic pathways in diverse symbioses

Annette Kampaa,1, Andrey N. Gagunashvilib,1, Tobias A. M. Guldera, Brandon I. Morinakaa, Cristina Daolioc, Markus Godejohannc, Vivian P. W. Miaod, Jörn Piela,e,2, and Ólafur S. Andréssonb,2

aKekule Institute of Organic Chemistry and Biochemistry, University of Bonn, 53121 Bonn, Germany; bFaculty of and Environmental Sciences, University of Iceland, 101 Reykjavik, Iceland; cBruker BioSpin GmbH, 76287 Rheinstetten, Germany; dDepartment of Microbiology and Immunology, University of British Columbia, V6T 1Z3 Vancouver, Canada; and eInstitute of Microbiology, Eidgenössische Technische Hochschule Zurich, 8093 Zurich, Switzerland

Edited by Nancy A. Moran, Yale University, West Haven, CT, and approved June 18, 2013 (received for review March 27, 2013) Bacteria are a major source of natural products that provide rich of mycobiont lectin genes (6), and PCR-based phylogenetics in opportunities for both chemical and biological investigation. Al- investigation of intrathalline bacterial diversity (7). though the vast majority of known bacterial metabolites derive In a number of bacterial– symbioses, bacterial from free-living , increasing evidence supports the wide- partners have been implicated in the production of complex spread existence of chemically prolific bacteria living in symbioses. A molecules derived from polyketide synthase (PKS) and nonri- strategy based on bioinformatic prediction, symbiont cultivation, bosomal peptide synthetase (NRPS) pathways (3, 8, 9). Exam- isotopic enrichment, and advanced analytics was used to character- ples include pederin, made by bacteria that live in rove beetles of ize a unique polyketide, nosperin, from a lichen-associated Nostoc the Paederus, and structurally related metabolites, the sp. cyanobacterium. The biosynthetic gene cluster and the structure onnamides and psymberin, produced by bacteria that live in of nosperin, determined from 30 μg of compound, are related to marine sponges (Fig. 2). In general, metabolites known or sus- those of the pederin group previously known only from nonphoto- pected to be of symbiont origin show remarkably low structural synthetic bacteria associated with beetles and marine sponges. The overlap with natural products discovered in screening programs presence of this natural product family in such highly dissimilar from free-living bacteria (10). This phenomenon raises the in- associations suggests that some bacterial metabolites may be spe- triguing question of whether symbiont chemistry might encom- fi ci c to symbioses with and encourages exploration pass structural scaffolds covering distinctive regions of chem- of other symbioses for drug discovery and better understand- ical space. ing of ecological interactions mediated by complex bacterial In this study, we applied a combination of metagenomic and metabolites. natural product discovery methods to identify nosperin, the first

biosynthesis | membranacea | trans-acyltransferase polyketide Significance synthase | 13C nuclear magnetic resonance

fi “ Remarkable chemical families are being recognized by studying ymbiosis, de ned by de Bary (1) as the living together of two fi ” diverse symbioses. We identi ed, through metagenomics, the Sorganisms, includes a broad range of partnerships, from fi – rst cyanobacterial trans-AT polyketide biosynthetic pathway loose associations to obligate interdependencies and para- in the Nostoc symbiont of the lichen site interactions. Many involve microbes, with perhaps the most and showed its expression in natural thalli. An isotope-based — successful between bacteria and early nucleated cells in the technique designed for characterizing minute amounts of ma- Precambrian—leading to mitochondria and chloroplasts in mod- terial confirmed predictions that its product, nosperin, is a dis- ern eukaryotes (2). Symbiotic interactions are being examined tinct member of the pederin family of compounds that was with increasing molecular detail, focusing not only on attributes previously thought exclusive to animal–bacteria associations. that may be beneficial for each individually but also on The unexpected discovery of nosperin in lichen expands the what might be important for the association. It is increasingly structural range and known distribution of this family of nat- being recognized that biosynthetic pathways leading to synthesis ural products and suggests a role associated with . of specialized metabolites may play key roles in the biology of symbiosis (3). Author contributions: A.N.G. and Ó.S.A. initiated project; A.K., A.N.G., V.P.W.M., J.P., and Ó.S.A. designed research; A.K., A.N.G., B.I.M., V.P.W.M., J.P., and Ó.S.A. performed re- are ancient and physiologically highly integrated search; A.N.G. carried out bioinformatic analyses of WGS, isolated Nostoc strains and symbioses between heterotrophic filamentous fungi (mycobionts) conducted gene expression studies; A.K. performed feeding studies and compound iso- and or coccoidal green (photobionts) that lations; A.K., T.A.M.G., B.I.M., C.D., and M.G. performed metabolic analyses and elucidated may date as far back as 600 Mya (4). The morphology of the structure; J.P. analyzed the trans-AT PKS genes and performed metabolic prediction; C.D. and M.G. contributed new reagents/analytic tools; A.N.G. and V.P.W.M. examined distri- characteristic and stable macroscopic body of a lichen, the bution of gene cluster; A.K., A.N.G., T.A.M.G., B.I.M., C.D., M.G., V.P.W.M., J.P., and Ó.S.A. , typically bears little resemblance to the individual analyzed data; A.K., A.N.G., T.A.M.G., V.P.W.M., J.P., and Ó.S.A. wrote the paper. organisms that form it and, in many cases, can be highly orga- The authors declare no conflict of interest. nized: fungal cells on the periphery for physical support and This article is a PNAS Direct Submission. MICROBIOLOGY protection and photobiont cells inside, providing photosynthate Data deposition: The sequences reported in this paper have been deposited in the Gen- or fixed nitrogen or both (5) (Fig. 1 A–C). Although the pho- Bank database [accession nos. GQ979609 (nsp gene cluster), JQ975876 (second trans-AT gene cluster), GU591312 (nostopeptolide-like gene cluster), JX181775 (P. membranacea tobionts can often be isolated in pure culture (Fig. 1D), most WGS Nostoc rRNA genes), KC489223 (heterocyst glycolipid gene cluster), KC291407 mycobionts (almost exclusively from the ) are re- (rbcLXS operon), and JX975209 (Nostoc sp. N6 rRNA genes)]. fractory to propagation in vitro by standard methods, and intact 1A.K. and A.N.G. contributed equally to this work. fi lichens cannot be maintained arti cially for long. Nevertheless, 2To whom correspondence should be addressed. E-mail: [email protected] or [email protected]. such limitations are gradually being overcome using advanced This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. analytical platforms, e.g., metagenomics in the characterization 1073/pnas.1305867110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1305867110 PNAS | Published online July 29, 2013 | E3129–E3137 Downloaded by guest on September 25, 2021 A B C D

Fig. 1. The Peltigera membranacea and Nostoc symbiont. (A) Lichen in situ. (Scale bar, 5 cm.) (B) Rhizines (Rhi) on lower surface and apothecia (Apo) protruding from thallus edge. (C) Thallus cross section illustrating stratified internal structure including photosynthetic layer (shown with arrows) between cortical and medullary mycobiont layers (above and below, respectively). (Scale bar, 100 μm.) (D) Nostoc sp. N6 in culture. (Scale bar, 100 μm.) (photograph for Fig. 1C, courtesy of Martin Grube).

member of the pederin family from a lichenized cyanobacterium of the Nostoc genome, such as hgl (involvedinheterocystgly- and a further example toward the emerging concept of symbio- colipid biosynthesis; SI Appendix,TableS1). The longer of the sis-associated natural product pathways (10). two gene clusters in P. membranacea, designated “nsp” (Fig. 3) had significant homology to the gene clusters for the biosynthesis Results of pederin family compounds and therefore was selected for Discovery of Trans-AT PKS Genes in the Lichen Metagenome. Peltigera further investigation. membranacea is a widely distributed terrestrial lichen carrying The nsp gene cluster consists of a 59-kb region with 3 large Nostoc sp. as its photobiont (Fig. 1 A–D). Total lichen DNA genes (nspA, nspC, and nspD) that encode multidomain PKS or extracted from field samples collected in Iceland processed for PKS/NRPS proteins and a suite of 10 smaller genes that encode whole genome sequencing (WGS) (11) revealed approximately accessory enzymes (Fig. 3; Table 1). The multidomain proteins equal contributions from the mycobiont, the photobiont, and the together comprise a “starter” module 0, followed by nine PKS or community of intrathalline microbes. Bioinformatic mining of PKS/NRPS elongating modules (modules 1–9). The 5′ end of the the initial metagenome assembly yielded 18 candidate clusters gene cluster, i.e., nspA (modules 0–3), nspB, and the beginning of containing genes that encode PKS enzymes (SI Appendix, Table nspC (module 4 and the KS region of module 5), as well as ac- S1). Among the putative bacterial gene clusters, two were cessory genes at the 3′ end of the cluster, have closely related members of the trans-acyltransferase (AT) PKS family (Fig. 3; SI counterparts in biosynthetic gene clusters of pederin-type com- Appendix, Fig. S1) in which AT domains are not encoded by the pounds (Fig. 3). The middle region, however, has primary affinities PKS genes but rather by a separate gene elsewhere: i.e., the ATs to NRPS–PKS biosynthetic pathways from other members of that load the polyketide building blocks are not integral parts of Proteobacteria or Cyanobacteria, viz., the end of nspC (modules the modules but act as free-standing units (10). This group of 5–7) is similar to the PKS genes of the rhizoxin (rhi) biosynthetic enzymes is particularly interesting because many of them are gene clusters from Burkholderia sp. (12) and Pseudomonas sp. responsible for products made specifically by symbiotic bacteria (13). Further downstream, the PKS genes have resemblances to (10). These gene clusters in the lichen are most likely derived gene clusters reported from various Nostocales or Oscillator- from the photobiont, as only Nostoc exhibited a high level clonal iales. An ∼3-kb region at the junction of nspC and nspD is es- presence, indicated by DNA sequence coverage in the WGS, and a pecially intriguing in bearing ∼80% identity at the DNA level to commensurate level of coverage was found for diagnostic markers a portion of the nos-like gene cluster (a cis-AT PKS pathway) in

Fig. 2. Pederin family compounds and symbioses. (Upper Left) Image of Paederus fuscipes courtesy of Christoph Benisch (www.kerbtier.de). (Upper Right) Image of Theonella swinhoei courtesy of Yoichi Nakao. (Lower Right) Image of Psammocinia aff. bulbosa adapted with permission from ref. 15. Copyright 2007 American Chemical Society. (Lower Left) Image of Mycale hentscheli courtesy of Mike Page.

E3130 | www.pnas.org/cgi/doi/10.1073/pnas.1305867110 Kampa et al. Downloaded by guest on September 25, 2021 PNAS PLUS

Fig. 3. Nosperin biosynthetic gene cluster nsp and flanking regions. Microsynteny and homology with pederin and onnamide biosynthetic gene clusters are indicated in gray. Similarity of nsp to other PKS biosynthetic gene clusters is indicated by double-headed arrows. Numbers denote individual modules. Genes with similar proposed functions (Table 1) are indicated with identical colors. β, genes involved in β-branch formation; T, transposon. See SI Appendix, Figs. S15 and S16 for details of regions flanking the nsp locus.

P. membranacea (SI Appendix, Fig. S2); a homolog of this cluster pathways with similar products can often facilitate prediction of in Nostoc sp. GSV224 is responsible for biosynthesis of nostopep- natural product structures generated by trans-AT PKSs (16, 19). tolide (14), a cyclic peptide-polyketide (SI Appendix,TableS2). When the Nsp KS sequences (KS1–KS9, referring to the module Altogether, the nsp locus appears to be an evolutionary mosaic of number in which the domain occurs) were aligned and compared trans-andcis-AT PKS fragments from diverse sources. with 494 homologs using KSs from cis-AT systems as an out- Expression of the nsp pathway was detected by RNA-seq group, the resulting clades were generally consistent with respect analysis in P. membranacea thalli freshly collected from the same to KS functions (SI Appendix, Fig. S3–S5). For example, all KSs location as the source material for the WGS. Consistent with with known function in the same group as KS1 accept acetyl expectations for a photobiont-specific gene cluster, nsp tran- starters incorporated by domains of the GCN5-related N-ace- scripts were observed in the main thallus tissue that contains tyltransferase family (GNAT) (20). In this way, partial structures both mycobiont and photobiont cells, but not in apothecia or for the substrates of KS1–3, 5, 7, and 9 were predicted (Table 2). rhizines, which are lichen structures that are derived only from As expected from the earlier analyses, KS1–5(nspA, nspC) were the mycobiont (Fig. 1B; SI Appendix, Table S3). Although trans- most similar to KSs of the pederin (21, 22) and/or onnamide (23) AT PKS systems have been found in a wide range of bacteria (16, PKS, and a full domain analysis revealed virtually complete ar- 17), none have been reported for cyanobacteria, which are oth- chitectural identity with corresponding portions of the ped and erwise rich sources of cis-AT PKSs (17, 18). These observations onn PKS–NRPS clusters over the first six modules, ending with suggested the possibility of metabolic products that might be KS5. The region also included an NRPS (module 4a) that cat- novel, not only from structural but also from ecological and alyzes the insertion of a glycine residue (Fig. 4). This observation evolutionary perspectives. indicated that a large part of the polyketide product would re- semble pederin and onnamides. The remainder of the core Prediction of the Compound Structure. Detailed examination of the structure was more difficult to predict, because two of the four ketosynthase (KS) domains in PKS gene clusters using phylo- KSs (KS6 and KS8) fell into clades consisting of KS0s, which are genetic methods and comparisons of module architecture in nonelongating KS variants that usually show little consistency

Table 1. List of the genes present in the nsp gene cluster and their predicted functions Protein Percent Accession ORF size Proposed function Closest homolog (protein,origin) identity number*

nspA 5,320 PKS PedI, Paederus fuscipes symbiont 42 AAR19304 nspB 371 Flavin-dependent oxygenase PedJ, P. fuscipes symbiont 66 AAR19305 nspC 8,252 PKS-NRPS OnnI, Theonella swinhoei symbiont 49 AAV97877 nspD 2,206 PKS JamP, Lyngbya majuscula 60 AAS98787 nspE 474 MatE efflux transporter SxtM1, Lyngbya wollei 54 ACG63829

nspF 285 O-Methyltransferase OnnH, T. swinhoei symbiont 57 AAV97876 MICROBIOLOGY nspG 86 ACP Cpap_1683, Clostridium papyrosolvens DSM 2782 58 EGD47495 nspH 411 β-Ketoacyl synthase Cpap_1682, C. papyrosolvens DSM 2782 60 EGD47494 nspI 420 HMG-CoA synthase PksG, Bacillus subtilis subsp. subtilis SC-8 72 EHA29460 nspJ 262 Enoyl-CoA hydratase Cpap_1678, C. papyrosolvens DSM 2782 52 EGD47490 nspK 442 Acyltransferase PedD, P. fuscipes symbiont 49 AAS47563 nspL 464 Cytochrome P450 PPSIR1_33239, Plesiocystis pacifica SIR-1 35 EDM78481 nspM 647 Asparagine synthase Acid_5610, Candidatus Solibacter usitatus Ellin6076 48 ABJ86557

*Accession numbers are for the GenBank database.

Kampa et al. PNAS | Published online July 29, 2013 | E3131 Downloaded by guest on September 25, 2021 Table 2. Analysis of KS domains present in the Nsp PKSs Domain Closest characterized relative (substrate specificity) Predicted specificity of KS clade Moiety present in nosperin

KS1 pederin KS1 (acetyl starter) Acetyl Acetyl KS2 onnamide KS2 (α-L-methyl + β-D-OH) α-L-methyl + β-D-OH α-L-methyl + β-D-OH (anti configured) KS3 onnamide KS3 (β-exomethylene) mostly β-exomethylene β-exomethylene KS4 onnamide KS4 (KS0)KS0 KS0 KS5 pederin KS5 (amino acid) amino acid glycine KS6 rhizoxin KS11 (KS0, double bond) KS0, double bond KS0, double bond KS7 rhizoxin KS12 (shifted double bond) shifted double bond shifted double bond KS8 bryostatin KS8 (KS0)KS0 KS0 KS9 oxazolomycin KS9 (serine) amino acid proline

between phylogeny and substrate structure (16). KS06 was po- encoding KS06; moreover, KS7, encoded by the module imme- sitioned in a small subclade containing homologs from the rhi- diately downstream, is highly similar to rhizoxin KS12, which zoxin and bacillaene PKSs that are involved in shifting double accepts a substrate with a shifted double bond (25). Together bonds from the α,β- to the β,γ-position (24, 25). These KSs are with the upstream NRPS module, these features strongly sug- found in modules harboring, in addition to the KS0 and the acyl gested the presence of an enamide moiety, which is not present carrier protein (ACP), a dehydratase (DH) domain postulated to in pederin or onnamides. KS9, associated with KSs elongat- catalyze double bond isomerization and characterized by a ing chains of amino acid residues, consistent with its position NSAF/YL instead of the usual DxxxQ/H motif involved in de- C-terminal to a second NRPS module (8a), was the only other hydration (26). The same elements are present in the nsp module KS with predictable function. An analysis of residues lining the

Apsn B C D E FG H I J K L M

III III translation

module 1 3 4a 6 8 9 0 2 4 5 7 8a

KR CR ? DH KR KR AT KR GN MT CR KR 0 A MT 0 DH MT A ER AT KS KS KS KS C KS KS KS KS0 C KS TE

S S S S S S S S S O O S O HO O O O O O O O HO HO HN MeO HO N O HO O O HO HN HN MeO O O N HO O HO O HO HN MeO MeO O O HO pederin orthology O HO MeO HN O O HO HN OH OH MeO MeO H MeO H O O N O N O OH O OH O NspM OH O OH O HO O O MeO N NH2 N OH O

NspL

OH MeO H Release O N OH O OH O O Nosperin N NH2

HO

Fig. 4. The nsp gene cluster, deduced architecture of the PKS proteins NspA, NspC, and NspD, and proposed biosynthesis of nosperin. GNAT, GCN5-related N-acetyltransferase family (20); KS, β-ketoacyl synthase; KR, ketoreductase; MT, C-methyltransferase; CR, crotonase superfamily (also known as enoyl-CoA hydratase) (30); KS0, nonelongating KS; C, nonribosomal peptide synthetase (NRPS) condensation domain; A, NRPS adenylation domain; DH, dehydratase; AT, acyltransferase; ER, enoyl reductase; TE, thioesterase; ?, unknown. Small black circles symbolize acyl and peptidyl carrier proteins. The positions of amplicons used for the nsp screening are shown with black boxes and roman numerals.

E3132 | www.pnas.org/cgi/doi/10.1073/pnas.1305867110 Kampa et al. Downloaded by guest on September 25, 2021 substrate pocket of the adenylation domain, known as the obtain insights into structural features of these compounds, the PNAS PLUS nonribosomal code (27, 28), returned a perfect match to pro- crude extract was subjected to repetitive HPLC-solid phase ex- line-activating domains (SI Appendix, Table S5). In the absence traction (SPE) purification with subsequent NMR analysis of of diagnostic downstream KS domains, the portions of the pol- target molecules eluted with fully deuterated solvent (SI Ap- yketide generated by modules 5, 7, and 9 were predicted using pendix,Figs.S6–S9). This method allowed collection of high- classical PKS colinearity rules (29), although they often apply quality 1H- and 13C-spectra, as well as COSY-, HSQC-, and poorly to trans-AT PKSs. These rules indicated the presence of HMBC-2D-NMR data from microgram amounts of the 13C- two additional methyl groups and a hydroxyl function. The ter- labeled material (SI Appendix,Figs.S8–S13). NMR signals minal elongation step was predicted to be catalyzed by module 9 characteristic of the predicted exomethylene, methoxy, and C- in NspD, a cis-AT PKS module with an integrated AT domain. methyl functions were detected for a component eluting at 30.3 This step remained obscure, because the module architecture min (SI Appendix, Figs. S6–S9), of which only 30 μg were (KS-AT-KR-ER-ACP-TE) contrasts with the canonical obtained. Further support for the identity of the compound came (KS-AT-DH-ER-KR-ACP-TE) and lacks a DH domain to pro- from ESI(+)-MS analysis, which indicated a multiply labeled minor vide the substrate for the subsequent enoyl reduction. This fea- component with an exact unlabeled mass of m/z = 564.2926, well ture suggested either that the ER domain is nonfunctional, within the predicted range and best fitting a calculated atomic despite the presence of key amino acid residues, or that the DH composition of C26H43N3O9Na (calculated: m/z = 564.2897). activity is provided in trans. MS/MS analysis of the molecular ion peak additionally revealed Further structural predictions were possible by comparison of a daughter ion consistent with the formal loss of MeOH (m/z = the accessory and post-PKS nsp genes to known pathways. The 532.2664; calculated for C25H39N3O8Na: m/z = 532.2635), sug- genes nspGHIJK resembled those typically involved in the gen- gesting a methoxy group in the predicted structure. Several other eration of polyketide β-branches (Table 1), indicating the pres- indicative fragments were also visible in the MS/MS data, e.g., ence of a pederin-type exomethylene bond (30). Because the an additional cleavage of an acetamide functionality (m/z = closest relatives of nspB and nspF in the ped and onn clusters 473.2260; calculated for C23H34N2O7Na: m/z = 473.2264). The encode an oxygenase and a methyltransferase (31), responsible, NMR data fully supported the identity of the compound as a respectively, for one oxygenation (at C7) and one methylation (at member of the pederin group and allowed elucidation of its the C6 acetal oxygen) within the corresponding moiety of ped- constitution. In combination with bioinformatic analysis it was erin, similar units in the nsp product were expected. The putative possible to predict most of the stereogenic elements, except the asparagine synthetase (NspM) and cytochrome P450 (NspL) configuration at C12 and C14 (Fig. 5; for a full description of the enzyme homologs, however, remained without counterpart in MS and NMR-based characterization see SI Appendix, Data S2). pederin-type pathways. The structure of the compound is in almost perfect agreement with the predicted features and represents a hybrid of pederin Isolation and Characterization of the Polyketide Nosperin. Using the and an unusual proline-containing terminal moiety not pre- preliminary structural information as a guide, total extracts of viously observed in this group of natural products. Two devia- whole lichens were examined for the presence of the predicted tions from the incomplete product prediction are the terminal metabolites. Due to copious amounts of diverse glycolipids and amide function, most likely generated by the asparagine syn- other metabolites, however, LC-MS and extensive NMR-guided thase-like protein NspM, and the hydroxyl moiety at C20, in- subfractionation failed to detect a pederin-type polyketide. As dicating post-PKS oxidation by the P450 homolog NspL. Nostoc symbionts have often been cultured from lichens (5), an Altogether, the data show that this compound, which has been alternative approach focusing only on the cyanobacterium was designated nosperin, represents a unique member of the taken. Macerated thalli of P. membrancacea wereplatedonBG-110, pederin family of natural products. a minimal medium lacking nitrogen, and cyanobacteria identifi- able as Nostoc sp. by microscopic examination were established Distribution of the nsp Locus in Cyanobacteria. Although the culti- in pure culture (Fig. 1D). The presence of the nsp cluster in three vated strain Nostoc sp. N6 carried the nsp cluster, it also random isolates was confirmed by PCR for amplicons repre- exhibited a distinctive 23S rRNA polymorphism and originated senting the PKS genes nspA and nspC, and the accessory gene, from a specimen of P. membranacea independent from that used nspF (Fig. 4; SI Appendix, Table S7). One strain, designated N6, in metagenome sequencing. These observations suggested that also characterized by sequencing of 16S and 23S ribosomal the nsp pathway might be common in P. membranacea photo- RNAs, was grown in BG-11 liquid medium for 4 wk to evaluate bionts or in Nostoc and possibly even other cyanobacteria. A gene expression in culture. Transcription of the nsp gene cluster PCR-based survey for nspA, nspC, and nspF indicated that the in Nostoc sp. N6 was confirmed by mapping RNA-seq data and nsp cluster was present in P. membranacea from several locations found to be fivefold higher than in the thallus, relative to ex- in Iceland, but samples of this lichen in British Columbia, Can- pression of rbcLXS,aNostoc reference marker (SI Appendix, ada, also included specimens where the targeted sequences were Table S3). When extracts were prepared from scaled up cultures, not detected (Table 3). Three nsp amplicons obtained from numerous metabolites were observed in small amounts. How- a specimen near Vancouver on the mainland were nearly iden- ever, due to the unusual architecture of the terminal Nsp tical (99.9%) to those from a Vancouver Island sample, but domains and the unknown nature of post-PKS modifications, showed ∼3% divergence from the Icelandic reference (SI Ap- prediction of the mass of the compound was challenging and pendix, Fig. S14). convincing candidates were not identified by MS analysis. A bioinformatic search for nsp-like sequences in GenBank (SI

In light of the challenges imposed by multicomponent trace Appendix, Table S2) and a PCR survey for amplicons of nspA, MICROBIOLOGY mixtures and the absence of a known mass, a strategy of stable nspC, and nspF in 26 cyanobacterial strains representing 15 isotopic enrichment followed by HPLC-SPE-NMR to address genera from all cyanobacterial orders (Materials and Methods) problems of sensitivity and complexity while allowing detection did not return any positive results except in the genus Nostoc, of predicted structural moieties was used. Nostoc sp. was cul- suggesting that the nsp pathway per se may have a phylogeneti- 13 tured in 25 L of BG-11 supplemented with C-labeled NaHCO3, cally restricted distribution. Within Nostoc there appeared to be and after 5 wk, cyanobacterial biomass from 10 L of culture was an association with lichens: results from PCR testing of four freeze-dried and extracted. HPLC-electrospray ionization (ESI)- strains (Nostoc sp. PCC9709, AR10B, AR9A, and WL-1) iso- MS analysis confirmed that most components in the extracts had lated from Peltigera spp. (32, 33) were positive for nsp, whereas multiple 13C atoms incorporated into individual molecules. To four other strains not associated with lichens (Nostoc muscorum

Kampa et al. PNAS | Published online July 29, 2013 | E3133 Downloaded by guest on September 25, 2021 OH OH O OH O 1 MeO H 20 O N 2 13 N NH2 10 O HO 24

Fig. 5. Stereochemical characterization of nosperin by NMR and bioinformatic analysis. The absolute configurations at the chiral centers were predicted by analysis of the stereospecificity of KR domains (blue), the NRPS domain structure (orange), the overall domain organization in comparison with other pederin- type biosynthetic gene clusters (red), and NMR coupling constants and/or chemical shifts (green).

PCC7906, Nostoc punctiforme PCC73102, Nostoc spp. PCC6705, trans-AT PKS–NRPS systems responsible for a number of animal– and PCC7107) were negative. bacteria symbiosis-associated compounds including pederin, the- The limited distribution within Cyanobacteria and the appar- opederins, onnamides, mycalamides, psymberin (irciniastatin A), ent absence of the nsp gene cluster in some samples from Can- and others (10) (Fig. 2). These compounds, with almost identical ada suggested that rather than being a core part of the genome, core regions but different biosynthetic starter regions and/or the nsp genes may have been introduced horizontally. Inves- termini, are often highly toxic to eukaryotes and some have been tigation of regions flanking the nsp cluster revealed IS4 elements considered promising candidates for anticancer drug de- on both sides and linkage to genes associated with plasmid velopment (38–40). Notably, this group of compounds has never replication and partitioning (including parA, parB,andparM been recovered from screening free-living bacteria, despite homologs and a gene encoding a DNA helicase), suggesting the conspicuous pharmacological activities. Studies of mycalamide A possibility of an extrachromosomal source (Fig. 3; SI Appendix, (Fig. 2), which binds in the E-site of the ribosome normally oc- Figs. S15 and S16). cupied by the tRNA-terminal CCA (41), and synthetic analogs together with molecular modeling, have identified the N-acyl Discussion linked tetrahydropyran structure as central to binding and ac- In this report, we describe identification of the nsp genes in the tivity (42). The presence of the N-acyl linked tetrahydropyran in P. membranacea lichen metagenome, the first trans-AT PKS gene nosperin suggests it might have similar bioactivity; however, the cluster from a cyanobacterium, and the application of a strategy amounts available were too small for testing. consisting of bioinformatic prediction, symbiont cultivation, iso- The discovery of nosperin not only increases the number of tope enrichment, and 13C-NMR that enabled characterization of chemical scaffolds and biosynthetic enzymes encompassed by the a unique symbiosis-associated natural product, nosperin, from pederin group but also expands the remarkable range of symbi- the photobiont. oses associated with this natural product family (Fig. 2). Fur- Lichens have long been known for distinctive mycobiont-pro- thermore, although the taxonomic identities of their producers duced compounds, such as depside and depsidone polyketides are unknown, with the exception of pederin, which is produced (34), but unique structures and pathways are now also emerging by a close relative of Pseudomonas aeruginosa (21, 43, 44), both from studies of the photobionts. Two conventional cis-AT PKS– lichen metagenomic data and expression and product charac- NRPS biosynthetic pathways have recently been described from terization in Nostoc sp. N6 clearly show that nosperin derives cyanobacteria associated with lichens: the mcy gene cluster (35, from the cyanobacterial photobiont of P. membranacea. Although 36) involved in synthesis of microcystins, notorious hepatotoxins this cyanobacterium is essential to every phase of thallus growth typical of many cyanobacteria, and the crp gene cluster, re- and development, it is also a facultative symbiont, being cultur- sponsible for production of cryptophycins (37), anticancer agents able by itself on basic mineral salts media. This first individually of more limited distribution. The elements of the nosperin bio- identified and culturable producer of a pederin family natural synthetic pathway, in contrast, are similar to the less common product provides new opportunities to study the biochemistry and

Table 3. Detection of nsp amplicons in P. membranacea from Iceland and two locations in British Columbia, Canada Region Locality Number of samples nspA nspC nspF

Reykjavík Grafarholt 1 + + + Keldur 3 + + + Mosfellsbaer 1 + + + Öskjuhlid 5 + + + Raudavatn 1 + + + Ulfarsfell 1 + + + Vancouver (Mainland) Belcarra 1 + + + Black Mountain 1 −−− Brothers Creek 1 + + + Eagle Ridge 2 + + + Eagle Ridge 3 −−− Vancouver Island Horth Hill 2 + + + Roche Cove 2 −−−

For positions of the amplicons see Fig. 4. +, a PCR product of the expected size was observed; −, no PCR product was observed.

E3134 | www.pnas.org/cgi/doi/10.1073/pnas.1305867110 Kampa et al. Downloaded by guest on September 25, 2021 physiology of the biosynthetic pathway in vivo, as well as to im- 13C-NMR–based technique and other recent methods such as im- PNAS PLUS prove metabolite yield through optimization of production pro- aging MS (53) can detect low concentration signatures of tocols and strain improvement. nosperin and facilitate investigation of its role in symbiosis. Study of genes, gene clusters, and biosynthetic pathways in These approaches could also identify molecular variants: e.g., diverse symbiotic associations may help clarify their functions or recently studied specimens of P. membranacea that appear identify metabolic products that are essential. Some polyketides negative for only one or two of the three primer sets used for nsp produced by trans-AT PKSs, such as pederin (21, 22, 43), screening may present variants of nosperin. This possibility is bryostatin (45, 46), and rhizoxin (12, 47), are known to partici- akin to the situation of the microcystins and cryptophycins for pate in host defense and pathogenicity in symbiotic associations. which a large number of structural variants have been found (36, It has also been suggested that PKS–NRPS compounds such as 37). A thorough study of the >1,500 of cyanobacteria- the microcystins, sometimes produced by cyanobacterial sym- bearing lichens and the multitude of other organisms including bionts, may contribute to the chemical defense of lichens against bryophytes, , cycads, and angiosperms (54) that harbor grazers (35, 36). Expression of the nsp genes in P. membranacea cyanobacterial symbionts may yield many new biosynthetic and their presence in all Icelandic specimens tested suggest that pathways and metabolites to provide both alternative chemistry it is a beneficial trait, although its role is unclear. In this regard, it for potential pharmacological applications and a wealth of in- may be significant that no microcystin pathway homolog was formation on the chemical biology of symbiosis. identified in P. membranacea, leaving open the possibility that nosperin might have a similar function in the lichen. Examina- Materials and Methods tion of geographically distant populations of P. membranacea Identification of PKS Gene Clusters in the P. membranacea Metagenome and was informative, as absence of nsp amplicons from some samples Expression Analysis of Whole Thalli. Metagenomic DNA was processed for from Canada indicates that these genes are not essential for the sequencing at commercial facilities via Roche 454 and Illumina Solexa 2 × 35-bp methodology generating 1.76 GB of 454 data and 1.4 GB of Illumina lichen symbiosis although nosperin may confer advantage under ∼ × some conditions. This provision may also apply to pederin, onna- data, yielding 50 coverage of the Nostoc genome. A draft assembly of the P. membranacea metagenome was constructed with MIRA v3.2.1 (www. mides, and psymberin, where the metazoan hosts can be found chevreux.org/projects_mira.html). To search for PKS gene clusters, concate- with or without the metabolites (23, 43, 48). In the case of nated consensus sequences of the KS (N terminus, pf00109; C terminus, P. membranacea, additional field studies may help elucidate pf02801; http://pfam.sanger.ac.uk/) and ACP domains (pf00698) were used in whether there is a primary cause, e.g., a founder effect, a par- a TBLASTN search (55) to retrieve all relevant contigs from the metagenomic ticular environmental condition, or an interplay of other factors database. Accuracy of the assembly was verified by visual inspection of the that underlie the distributional differences observed. contigs in GAP4 (Staden package) (56) based on a mapped 3.5-kb paired-end The presence of similar trans-AT PKS–NRPS gene clusters in library. Portions of the nsp sequence were verified by PCR amplification and sequenced directly using BigDye chemistry (Applied Biosystems; MacroGen). different groups of bacteria has suggested that these clusters are fi horizontally transferred (44). The flanking of the nsp cluster by RNA-seq data sets from eld samples of lichen thalli, apothecia, and rhizines were previously generated (6) and used for mapping in this study with transposable elements is consistent with this hypothesis, and the Bowtie (57). mosaic of homologies across the gene cluster suggests involve- ment of several intergenomic and intragenomic recombination Structure Prediction. Amino acid sequences of 503 KS domains from trans-AT events. The homology of NspE and part of NspD to proteins and cis-AT PKSs were retrieved from GenBank and aligned using the MUSCLE from Oscillatoriales (Fig. 3) suggests that an ancestral ped-like algorithm with a gap open score of −1, as implemented in Geneious 5.5.3 operon, specifying the conserved core part the molecule, may (Biomatters Ltd.). After manual improvement of the alignment, phyloge- have been introduced into and modified by oscillatorean cya- netic reconstruction was performed by means of the Geneious software nobacteria: the position of nspE and nspD between sequences using the neighbor joining algorithm with a Jukes-Cantor distance method. with high homology to the ped gene cluster and the orientation of KS domains of cis-AT PKSs were used as an outgroup. Bootstrap analysis was nspE opposite to the ped-like genes suggest an intragenomic done with 1,000 pseudoreplicate sequences. rearrangement mediated by genetic similarity of PKS–NRPS Chemical Analysis of Whole Lichen. Air-dried lichen (30 g) was ground to a fine modules. Transfer to Nostocales and subsequent recombination ∼ powder in liquid nitrogen using a mortar and pestle and stirred for 24 h at resulted in the present domain organization that includes the 3-kb room temperature in MeOH. The mixture was filtered, and the solid material cis-AT containing fragment from a Nostoc nos-like cluster. The was extracted a second time. The solvent of the combined MeOH extracts was presence of a cis-AT domain is unusual in a PKS relying on removed under reduced pressure. The crude extract was partitioned between

trans-ATs, with few occurrences among the ∼40 large trans-AT 10:1 MeOH/H2O (300 mL) and n-hexane (3 × 100 mL). The solvent was re- PKS complexes with known products (10, 49). A relatively recent moved from the aqueous MeOH layer under reduced pressure, and the insertion of this ∼3-kb fragment is indicated by the high amino residue was further fractionated by silica gel column chromatography. The acid and nucleotide similarities to the nos cluster and suggests following solvents (0.5 L each) were used to elute compounds: petroleum that near relatives of the nsp pathway may exist. It will be in- ether, petroleum ether/EtOAc (1:1), EtOAc, EtOAc/MeOH (9:1; 8:2; 7:3, 1:1), and MeOH. The fractions were evaporated under reduced pressure and teresting whether a gene cluster similar to nsp, but without the analyzed by LC-MS using a Phenomenex Luna C18 column with a mobile cis-AT encoding region, or with other types of inserts and sub- phase gradient of 1:9 CH3CN/H2O + 0.1% TFA to 100% acetonitrile over stitutions, will be found in other bacteria. Study of such examples 30 min and a flow rate of 1 mL/min. of naturally engineered multidomain genes and gene clusters involving distantly related participants may not only generate Isolation of Nostoc sp. N6. Lichen thalli collected from the same location as useful hypotheses for further understanding their evolution, but material used for WGS and expression studies were macerated between sterile microscope slides (58), and cells were plated on BG-110 (without the phylogenetic reconstruction may also be informative in MICROBIOLOGY identifying models of successful architectures for application in NaNO3) agar medium (59) and incubated at 20 °C with a 12/12-h day/night combinatorial biosynthesis industrially. cycle. Nostoc colonies were purified by repeated streaking on the same The metabolic options offered by symbiotic associations pro- medium and maintained at room temperature. Analysis of the 16S and 23S rRNA sequences in the RNA-seq library (below) confirmed both the purity of vide exciting potential for drug development and highlight the the culture and its identification as a Nostoc sp. need for new discovery strategies applicable to these complex systems. Although individual steps of the present procedure have RNA Extraction and RT-PCR of Nostoc sp. N6. Total RNA was isolated from 1 L of been used previously in natural product research (15, 19, 47, BG-11 medium incubated at 20 °C under constant illumination for 4 wk. 50–52), the combination of methods has not been reported Cyanobacteria were retained on Miracloth (Calbiochem) after culture fil- and should be applicable to many further organisms. The tration, rinsed with , blotted with paper towels, flash frozen in liquid

Kampa et al. PNAS | Published online July 29, 2013 | E3135 Downloaded by guest on September 25, 2021 nitrogen, and crushed to a fine powder. TRIzol reagent (Life Technologies) heteronuclear multiple-bond correlation spectroscopy (HMBC) were carried was added to the powder, and it was ground again. The mixture was out using 4,000 complex data points in F2 and 512 points in the F1 di- transferred to a 15-mL polypropylene tube and processed according to the mension. The multiplicity edited gradient heteronuclear single quantum TRIzol protocol. Before RT-PCR, the RNA was treated with DNase I (RNase- correlation (HSQC) was acquired with 2,000 data points in F2 and 400 points free) (Fermentas) to remove residual genomic DNA. First-strand cDNA was in the F1 dimension. The COSY experiment was acquired with 32 scans, the synthesized from 1 μg total RNA using SuperScript II Reverse Transcriptase HSQC with 64 scans, and the HMBC with 128 scans per increment, resulting in (Invitrogen). RNA-seq data were obtained using Illumina Solexa Genome experiment times of 8 h 46 min (COSY), 12 h 4 min (HSQC), and 1 d 11 h Analyzer IIx at the deCODE Genetics facility (Reykjavik, Iceland). RNA-seq (HMBC). A C13 spectrum with composite pulse decoupling on the proton mapping was done with Bowtie (57) and visualized in Geneious 5.5.3. channel was acquired by collecting 4,096 scans with 131,072 complex data points at a sweep width of 40,761 Hz and with a relaxation delay of 5 s. The Culture of Nostoc sp. N6 for Natural Product Analysis. Twenty-five liters of cells experiment time was 7 h 11 min. were grown in an illuminated (5,200 lm) bubble-column bioreactor in BG-11 13 liquid medium, optionally enriched with 3 mM C-labeled NaHCO3,for5wk Distribution Survey. P. membranacea thalli were collected at several localities at pH 7.8 and 25 °C. The cyanobacteria from 10- and 5-L portions of the in Iceland (Reykjavik area) and in British Columbia (North Shore mountains fi culture were collected by ltration, frozen in liquid nitrogen, freeze-dried, near Vancouver; Vancouver Island), and DNA was extracted using the pre- and stored at −20 °C. viously described methods (60). DNA samples representing cyanobacterial strains other than those newly isolated from P. membranacea for this study Nostoc Chemical Extractions and Analysis of sp. N6, Unlabeled Culture. Freeze- were prepared and described previously (32, 61) and stored at −20 °C. They dried cyanobacteria (above) were homogenized in 50 mL CH Cl /MeOH (2:1) 2 2 include Anabaena sphaerica UTEX1616, Chlorogloeopsis fritschii PCC6718, and stirred for 15 min at room temperature. Biomass was filtered and Cylindrospermum stagnale PCC7417, Fischerella muscicola PCC7414, treated again with the same amount of CH Cl /MeOH for 30 min at 30 °C. 2 2 Geitlerinema sp. PCC7105, Gloeobacter violaceus PCC7421, Leptolyngbya sp. This procedure was repeated twice. The combined extracts were dried under PCC7104, Leptolyngbya sp. PCC7375, Lyngbya kuetzingii UTEX1547, reduced pressure. The crude extract was dissolved in MeOH and subjected to Myxosarcina sp. PCC7325, Nodularia spumigena PCC73104, Nodularia LC-MS analysis using an Agilent 1200 series HPLC and Bruker Daltonics harveyana UTEX2093, Pleurocapsa sp. PCC7315, Pleurocapsa sp. PCC7324, micrOTOF-Q-spectrometer. HPLC was carried out with a Phenomenex Luna Pleurocapsa sp. PCC7321, hofmanni PCC7110, Synechocystis C18 column (5 μm, 250 × 2.00 mm), a mobile phase gradient of CH3CN/H2O PCC6803, Nostoc punctiforme PCC73102A, Nostoc sp. PCC6705, Nostoc sp. (20:80) to (80:20) over 45 min, and a flow rate of 1 mL/min. PCC9709, Nostoc sp. AR10B, and Nostoc sp. AR9A. In addition, DNA samples from Calothrix sp. PCC7601, Nostoc muscorum PCC7906, and Nostoc sp. Chemical Extractions and Analysis of Nostoc sp. N6, Labeled Culture. Freeze- PCC7107 (originally obtained from the Pasteur Culture Collection of Cya- dried cyanobacteria were extracted with stirring for 24 h in 2 L MeOH at room temperature. After filtration, the methanolic fraction was dried by evapo- nobacteria) and Nostoc sp. WL-1 (kindly provided by E. Loos, University of ration and redissolved in 0.5 L MeOH/H O (10:1) followed by liquid-liquid Regensburg, Regensburg, Germany) were prepared from cultures using 2 fi extraction with 0.5 L cyclohexane. The cyclohexane fraction was discarded. similar methods (32). Ampli cation of rbcLX (62) or rnpB (63) regions (SI Appendix, Table S7) (and in some cases, also the 16S rRNA gene) was used as The remaining MeOH/H2O fraction was dried and stored at −20 °C. This material was directly used for LC-SPE-NMR analyses. a positive control to ensure DNA quality before screening with primer sets targeting the nsp gene cluster (Fig. 4). DNA from the Nostoc sp. N6 strain HPLC-SPE-NMR. The solvent system consisted of eluent A (H O + 0.1% deu- was used as an nsp positive control. Conditions for nsp screening were 94 °C 2 × terated formic acid) and eluent B (acetonitrile) with a linear gradient starting for 2 min, then 94 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s (35 ), and then with 10% of B up to 90% B in 30 min. The flow rate was 0.8 mL/min at 25 °C, 72 °C for 7 min. For rbcLX primers, the extension time was 1 min. Eppendorf and the injection volume was 50 μL. The chromatography was monitored at MasterMix 2.5× (Eppendorf) was used according to the manufacturer’s 210, 220, and 254 nm, and these wavelengths were used to define absorbance protocol in a final volume of 50 μL. All negative samples were repeated at

thresholds to trigger SPE trapping. The HPLC eluate was diluted with H2O least once. PCR amplicons from two samples were sequenced directly (2.4 mL/min) before trapping on SPE cartridges (Spark Holland), and indi- (MacroGen). The bioinformatic search was conducted in November 2012. vidual peaks were trapped four times to increase concentration on cartridge. The cartridges were dried with pressurized nitrogen gas for 30 min each, and ACKNOWLEDGMENTS. We thank T. Taylor and W. Loos for a gift of lichens

the analytes were eluted with 190 μLCD3CN (99.8 atom %; Deutero GmbH) and Nostoc isolate WL-1; K. Anamthawat-Jónsson for help with ; into 3-mm match tubes from Bruker BioSpin GmbH. deCODE Genetics (D. N. Magnúsdóttir, G. P. Örlygsdóttir, S. Snorradóttir, and Ó. T. Magnússon) for sequencing; G. König for providing a fermentor; H. Gross for sharing knowledge on culturing cyanobacteria; and K. Peters- NMR. All NMR experiments were acquired on an AVANCE III 600 MHz NMR Plaumbaum and M. Engeser for MS support. This work was financially sup- spectrometer equipped with a 5-mm QNP cryo probe head (Bruker Biospin). ported in part by the DFG (SFB 642 to J. P. and Emmy Noether fellowship to Standard parameter sets created for the Bruker SELU (structure elucidation) T.A.M.G.), the EU (BlueGenics to J.P.), the Alexander von Humboldt Founda- program were uniformly used. Gradient correlation spectroscopy (COSY) and tion (B.I.M.), and the Icelandic Research fund (to Ó.S.A.).

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