RESEARCH ARTICLE SUMMARY◥

SYMBIOSIS

A microbial factory for defensivekahalalides in a tripartitemarine symbiosisJindong Zan*, Zhiyuan Li*, Ma. Diarey Tianero, Jeanette Davis,Russell T. Hill, Mohamed S. Donia†

INTRODUCTION: Chemical defense strate-gies, in which organisms use toxic moleculesfor protection against pathogens or predators,are widespread in themarine environment. Insome cases, the same defensive molecules areshared by taxonomically distant organisms,raising questions about their molecular origin.The actual source of these molecules may bethe organism itself, as observed inmarine algae; a microbial symbiont,as commonly seen inmarine spongesand tunicates; or diet, as in severalmarine mollusks. Elucidating themolecular basis of toxin productionin chemically defended organisms isimportant for a complete understand-ing of their ecological interactions.

RATIONALE: Inthiswork,westudiedtoxin production in the Hawaiianmarine algaBryopsis sp. and its pred-ator, the mollusk Elysia rufescens.Both organisms are chemically de-fended against predators by a di-verse library of lipopeptide toxins,the kahalalides, but the details ofkahalalide production and diversifi-cation are unknown (see the figure).One of thesemolecules, kahalalide F,is a potent cytotoxin and has beenevaluated clinically as an anticanceragent. The molecular structuresof the kahalalides show several fea-tures of microbial biosynthesis:They are fatty acid–cyclic peptidehybrids with several D- and non-proteinogenic amino acids, thus motivatingus to hypothesize that the kahalalides areproduced by a bacterial or fungal symbiontof Bryopsis sp. or of both Bryopsis sp. andE. rufescens. We combined metagenomic,metatranscriptomic, and chemical analyseswith microbial cultivation, fluorescence mi-croscopy, and evolutionary genomics to de-termine the molecular bases of kahalalideproduction and evolution in this tripartitemarine symbiosis.

RESULTS:Using metagenomic analyses, wediscovered a bacterium—termed “CandidatusEndobryopsis kahalalidefaciens”—that has nocultured close relatives, andwe show that it livesin symbiosis with the alga Bryopsis sp. (see thefigure). Using fluorescencemicroscopy, bacterialcultivation, and comparative genomics,we showthat “Ca.E. kahalalidefaciens” is an intracellular,

obligate, genome-reduced symbiont that has lostessential functions for free living (e.g., amino acidbiosynthesis). Despite this reduced metaboliccapacity, 20% of the “Ca. E. kahalalidefaciens”genome encodes a diverse set of 20 nonribo-somal peptide synthetase pathways. We linknine of these pathways to nine structurallydiverse kahalalides (including kahalalide F,the main defensive toxin of both Bryopsis sp.and E. rufescens), which we then chemicallyidentify in the same sample of Bryopsis sp.

None of the amino acid substrates thatmakeup the kahalalides can be produced by “Ca.E. kahalalidefaciens” itself; therefore, thesesubstances are mostly provided by the auto-trophic Bryopsis sp., highlighting an unusualstrategy of collaborative biosynthesis be-tween a symbiotic bacterium and its host.Moreover, using metagenomic analysis andfluorescence microscopy, we show that “Ca.E. kahalalidefaciens” is not a symbiont of the

mollusk E. rufescens, es-tablishing chemical seques-tration as the means bywhich this animal indi-rectly acquires bacteriallyproduced kahalalides fromits algal diet.

Detailedanalysisof the“Ca.E.kahalalidefaciens”genome reveals a high level of plasticity anda distinctive model of diversifying evolutionthat is independent of horizontal gene trans-fer, consistent with the intracellular lifestyleof this symbiont. In this model, new non-ribosomal peptide synthetase pathways arisethrough duplication and divergence, accom-

panied by extensive interpathwayrecombination events. Finally, meta-transcriptomic analysis reveals that26% of the transcriptional activity of“Ca. E. kahalalidefaciens” is dedi-cated to kahalalide biosynthesis andthat biosynthetic pathways for dif-ferent kahalalides vary widely intheir expression levels, further em-phasizing the importance of thesemolecules in maintaining a success-ful symbiosis.

CONCLUSION: In a chemically de-fended, tripartite marine symbiosis,we show that an obligate bacterialsymbiont of a marine alga producesa library of defensive molecules thatprotect the host from predation,and that the same molecules arein turn hijacked by a predatory mol-lusk and used for its own defense.Living intracellularly in algal cells,the symbiont acts as a microbial fac-tory for the biosynthesis of complexdefensive molecules from simplehost-derived substrates. Symbiont-derived production of defensive mol-

ecules in marine algae and indirect acquisitionof microbial products by predatory mollusksmay thus be important yet rarely studiedphenomena in marine ecosystems.▪

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Zan et al., Science 364, 1056 (2019) 14 June 2019 1 of 1

The list of author affiliations is available in the full article online.*These authors contributed equally to this work.†Corresponding author. Email: donia@princeton.eduCite this article as J. Zan et al., Science 364, eaaw6732(2019). DOI: 10.1126/science.aaw6732

Kahalalide toxins

Ca. E. kahalalidefaciens

Bryopsis sp.

E. rufescens

Chemical defense in a tripartite marine symbiosis.Thebacterial symbiont “Ca. E. kahalalidefaciens,” which lives intra-cellularly in the marine alga Bryopsis sp., produces a diverselibrary of toxins (the kahalalides) that protect the host frompredation. The mollusk E. rufescens sequesters the same toxinsfrom its algal diet and employs them for its own defense.

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RESEARCH ARTICLE◥

SYMBIOSIS

A microbial factory for defensivekahalalides in a tripartitemarine symbiosisJindong Zan1*, Zhiyuan Li2,3*, Ma. Diarey Tianero1, Jeanette Davis4†,Russell T. Hill4, Mohamed S. Donia1‡

Chemical defense against predators is widespread in natural ecosystems. Occasionally,taxonomically distant organisms share the same defense chemical. Here, we describe anunusual tripartite marine symbiosis, in which an intracellular bacterial symbiont(“Candidatus Endobryopsis kahalalidefaciens”) uses a diverse array of biosyntheticenzymes to convert simple substrates into a library of complex molecules (the kahalalides)for chemical defense of the host, the alga Bryopsis sp., against predation. Thekahalalides are subsequently hijacked by a third partner, the herbivorous molluskElysia rufescens, and employed similarly for defense. “Ca. E. kahalalidefaciens” has lostmany essential traits for free living and acts as a factory for kahalalide production.This interaction between a bacterium, an alga, and an animal highlights the importanceof chemical defense in the evolution of complex symbioses.

One of the most efficient defense strate-gies in the ocean is chemical defense, inwhich organisms accumulate toxic smallmolecules that kill and/or deter predators(1). Because of the structural complexity

of these defensive chemicals and their similarityto bacterial products, many have been proposedto originate from bacterial symbionts (2, 3). In-deed, defensive molecules of substantial toxicityhave been genetically linked to specific bacterialsymbionts of several marine animals (4). Theconcept of chemical defense can also extendbeyond a single animal, with the same chem-icals benefiting a network of partners. Thisphenomenon is common in marine mollusksthat prey on chemically defended organismsand sequester the organisms’ toxic chemicalsfor their own defense (5). Although these twophenomena—production of defensive chem-icals by bacterial symbionts and sequestrationof defensive chemicals by predatory mollusks—have been separately described in marine sys-tems, their integration in multipartite symbiosesis rarely studied.In this work, we investigated the marine alga

Bryopsis sp. and its predator mollusk Elysia

rufescens (Fig. 1). Bryopsis sp. harbors a toxiclipopetide molecule, kahalalide F (KF) (6), whichwas shown to be responsible for chemical de-fense against predators (7). Unlike other pred-ators that are repelled by this toxin, E. rufescensnot only feeds on KF-containing Bryopsis sp.,but it also accumulates KF in its body at severaltimes the concentration in Bryopsis sp. andemploys KF for its own defense (7–9). Similarlyto other species of Elysia, E. rufescens addition-ally maintains photosynthetically active algalchloroplasts in its digestive organs for severalmonths, in a phenomenon known as kleptoplasty(10). Although KF has been isolated only fromBryopsis sp. and E. rufescens, it contains struc-tural features that suggest a possible microbialorigin, namely nonproteinogenic amino acids(e.g., ornithine, dehydrobutyrine, and severalD-amino acids) and a fatty acid moiety (Fig. 1).These features motivated us to hypothesize thatKF is produced by a cryptic third partner, suchas a bacterial or fungal symbiont. To test thishypothesis and study the molecular and evolu-tionary details of toxin production in this un-usual system, we employed a multidisciplinaryapproach that combines metagenomic, meta-transcriptomic, and chemical analyses; micro-bial cultivation; fluorescence microscopy; andevolutionary genomics.

KF production by a bacterial symbiont

We collected fresh Bryopsis sp. specimens froman algal bloom at the exact location where KF wasoriginally reported (Black Point Bay, Honolulu,Hawaii; sample designated Bryopsis-2015) (Fig.1A) and analyzed them using a variety of chem-ical and molecular techniques. First, we chem-

ically extracted Bryopsis-2015 as previouslydescribed (6), analyzed its resulting organicextract using high-performance liquid chro-matography coupled with high-resolution tandemmass spectrometry (HPLC–HR-MS/MS), and con-firmed that it harbors KF (fig. S1 and table S1).Second, to investigate the microbial commu-nity associated with Bryopsis-2015, we usedhigh-throughput 16S ribosomal RNA (rRNA)gene amplicon sequencing (104,000 reads, onaverage, V4 region) on DNA isolated from mul-tiple replicates of the same algal collection. Sur-prisingly, only three bacterial classes dominate theBryopsis-2015 microbiome—Gammaproteobacteria(11.8% of the reads, on average), Alphaproteo-bacteria (9.3%), and Flavobacteriia (11.8%)—whereas most of the remaining reads (57.0%)map to the chloroplast-encoded 16S rRNA gene inBryopsis sp. (Fig. 1C). A single sequence accountsfor 74.1 to 92.0% of the fraction assigned to theclass Flavobacteriia, making it the single mostdominant species inBryopsis-2015.Notably, fourbiological replicates collected on two consecutivedays showed consistent microbiome profiles.These results reveal a relatively simple and uni-form microbiome composition for KF-containingBryopsis sp.Owing to the cyclic lipopeptide nature of KF

(Fig. 1D), this compound could be synthesizedeither by a nonribosomal peptide synthetasepathway (NRPS) or by a ribosomally synthesizedand posttranslationally modified peptide path-way (RiPP) (11, 12). In both cases, it is relativelystraightforward to computationally link a bio-synthetic gene cluster to its product. In NRPSs,the number of modules, the substrate specificityof the adenylation (A) domains, and the pres-ence or absence of epimerization (E) domainsare distinctive for a particular molecule. In RiPPs,the primary amino acid sequence of the finalmolecule is directly encoded on a precursor pep-tide within the biosynthetic gene cluster. Toexamine whether bacterial or fungal membersof the Bryopsis sp. microbiome encode biosyn-thetic gene clusters consistent with the structureof KF, we deeply sequenced the Bryopsis-2015metagenomic DNA using Illumina [48 millionpaired-end reads, 175 base pairs (bps)], per-formed multiple iterations of assemblies on theproduced data, and queried the reads and re-sulting assemblies for possible biosynthetic geneclusters using several search strategies (see ma-terials and methods).First, for RiPP pathways, we searched for the

expected primary amino acid sequence of KF(VTVVPRITIVFTV,whereR replaces ornithine andT replaces dehydrobutyrine) against a databaseof algal metagenomic reads and contigs usingthe tBLASTnmode of the Basic Local AlignmentSearch Tool. Second, for NRPS pathways, weconstructed a database of 36 phylogeneticallydiverse NRPS proteins and used it as a query fortBLASTn searches against the Bryopsis-2015metagenomic assembly (18,308 scaffolds withlength >5 kbps). Third, we subjected the same scaf-folds to antiSMASH analysis, a stand-alone toolfor the unbiased identification of small-molecule

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Zan et al., Science 364, eaaw6732 (2019) 14 June 2019 1 of 10

1Department of Molecular Biology, Princeton University,Princeton, NJ 08544, USA. 2Princeton Center for TheoreticalScience, Princeton University, Princeton, NJ 08544, USA.3Center for the Physics of Biological Function, PrincetonUniversity, Princeton, NJ 08544, USA. 4Institute of Marineand Environmental Technology, University of Maryland Centerfor Environmental Science, Baltimore, MD 21202, USA.*These authors contributed equally to this work. †Present address:National Marine Fisheries Service, National Oceanic and Atmo-spheric Administration, Silver Spring, MD 20910, USA.‡Corresponding author. Email: donia@princeton.edu

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biosynthetic gene clusters (13, 14). Althoughno matches to the primary KF sequence wereretrieved using the RiPP search strategy, 130scaffolds (5 to 394 kbps in length) that encodeNRPS pathways were discovered using the NRPSsearch strategy. In addition, antiSMASH anno-tated a total of 25 scaffolds as NRPSs, only threeof which were not detected with the BLASTstrategy. Unfortunately, none of the 133 scaf-folds containing possible NRPSs constituteda 13-domain pathway, as would be expectedfor KF, implying that the presumed KF bio-synthetic gene cluster may be fragmented orunclustered. Careful analysis of themultimodular-NRPS–containing scaffolds revealed 33 scaffoldsthat have similar average GC content (~50%) andsimilar coverage in the metagenome (>300X),suggesting that they may be part of one largeNRPS pathway or at least part of the same bac-terial genome (fig. S2).To obtain a better assembly for the fragmented

NRPS scaffolds, we subjectedBryopsis-2015meta-

genomic DNA to long-read, single-molecule real-time (SMRT) sequencing using the Pacific Bio-sciences platform (three SMRTcells, 325,000 reads,median insert size of 9 kbps). After assembly anderror correction with the high-quality Illuminareads, we successfully closed a ~2.3-Mbp bacte-rial genome in which several of the partial NRPSsequences from the Illumina assembly were joinedto produce a 55.6-kbp NRPS pathway (NRPS-8)that is consistent with the final KF structure(Fig. 2). Several lines of evidence link NRPS-8to KF biosynthesis: (i) It begins with a startingcondensation domain, as predicted by multiplebiosynthetic analysis algorithms (NaPDoS andantiSMASH); these types of domains catalyzean amide bond formation between a fatty acidand the first amino acid of the growing peptidechain (13–15). Indeed, the KF structure startswith 5-methyl hexanoic acid conjugated to thefirst valine residue. (ii) It consists of 13 NRPSmodules, as expected for the tridecapeptide KF.(iii) Modules 1, 4, 5, 7, 8, 9, 10, and 12 of NRPS-8

contain epimerization domains, which are re-sponsible for converting the stereochemistry ofthe loaded amino acid from the L form to theD form (11). In agreement with this, KF harbors7 D-amino acids at positions 1, 4, 5, 7, 8, 9, and 10,which correspond exactly to these epimerizingmodules (threonine residue at position 12 is de-hydrated to dehydrobutyrine, which is achiral).(iv) The predicted substrate specificity of almostall adenylation domains in NRPS-8 matches theamino acid residues observed in KF, in an anal-ysis performed using published algorithms andfurther supported by a detailed phylogeneticanalysis (see materials and methods and figs. S3and S4) (16). Notably, at 40 kbps downstream ofNRPS-8, the same genome encodes a 16S rRNAgene that is identical to the aforementioned,most abundant bacterium in the sample. A 16SrDNA-based phylogenetic tree of related organ-isms placed this bacterium as a previously un-known genus in the family Flavobacteriaceae,order Flavobacteriales, class Flavobacteriia, and

Zan et al., Science 364, eaaw6732 (2019) 14 June 2019 2 of 10

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Fig. 1. Bacterial composition and chemical defense of the Bryopsis–Elysia symbiotic system. (A) The natural Bryopsis sp. bloom studiedin this work. (B) E. rufescens feeding on Bryopsis sp. in the laboratory.(C) Composition of Bryopsis-2015–associated bacterial communitiesacross four different replicates collected on two consecutive days (A andB: 29 March 2015; C and D: 30 March 2015) and grouped at the class

level. Rare taxa (<1% of total sequences) are labeled as “Others.” The insetbar graph represents the percentage of a single 16S rDNA sequence(hereafter designated “cEK”) that dominates the class Flavobacteriia.(D) Molecular structure of kahalalide F (KF), the main defensive chemicalin both Bryopsis sp. and E. rufescens. Structural features commonlyassociated with microbial biosynthesis are depicted in red, blue, and pink.

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phylum Bacteroidetes (fig. S5)—a taxonomicalgroup not known as a prolific producer of com-plex small molecules. We termed this bacterium“Candidatus Endobryopsis kahalalidefaciens”(Latin for “kahalalide-making”).

Symbiont biosynthetic capacity

Further analysis of the “Ca. E. kahalalidefaciens”genome revealed that it harbors 20 NRPS path-ways, ranging in size from 3.9 (NRPS-4) to 55.6(NRPS-8) kbps and accounting for all 33 high-coverage, ~50% GC NRPS fragments previouslyfound in the Illumina assembly (Fig. 2 and fig. S2).Unusually for bacteria, these NRPS pathways oc-cupy 20% of the genome. Since the initial dis-

covery of KF from Bryopsis sp. and E. rufescens,other work has detected >15 cyclic and linearlipopeptides of different lengths and amino acidcompositions from samples collected at the samesite; these molecules are known collectively asthe kahalalides (Fig. 3) (8, 9, 17–20). Becausethese molecules have been reported from thesame species (sometimes at abundances sim-ilar to that of KF) and because of the structuralsimilarities between KF and the rest of thekahalalides, we asked whether the remaining19 NRPSs in the “Ca. E. kahalalidefaciens” ge-nome encode other known kahalalides.To answer this question, we compared the

19 remaining NRPSs encoded in the genome

to the kahalalide structures. Overall, we wereable to link eight additional NRPSs in the “Ca.E. kahalalidefaciens” genome to eight corre-sponding kahalalide chemotypes (a distinctchemotype can encompass several kahalalidessharing the same amino acid sequence anddiffering only in the length or hydroxylation ofthe fatty acid, hydroxylation of a proline residuein the molecule, or linearization of the cyclicpeptide) (Fig. 3 and fig. S6). Although a geneticknockout (loss of function) or heterologous ex-pression (gain of function) is typically neededto unequivocally connect biosynthetic gene clus-ters to their products, five lines of evidence in-dicate that “Ca. E. kahalalidefaciens” synthesizes

Zan et al., Science 364, eaaw6732 (2019) 14 June 2019 3 of 10

Fig. 2. The large biosyntheticcapacity of “Ca. E. kahalalidefaciens.”(A) Circular map of the “Ca.E. kahalalidefaciens” chromosome.Tracks (from outermost to innermost)represent genes on the forward frame,genes on the reverse frame, RNAs,GC content, and GC skew. Genes arecolor coded according to the Cluster ofOrthologous Groups (COG) categories inthe Integrated Microbial Genome plat-form (21) (A, RNA processing and mod-ification; B, chromatin structure anddynamics; C, energy productionand conversion; D, cell cycle control,cell division, and chromosomepartitioning, E, amino acid transport andmetabolism; F, nucleotide transportand metabolism; G, carbohydratetransport and metabolism; H, coenzymetransport and metabolism; I, lipid transportand metabolism; J, translation,ribosomal structure, and biogenesis;K, transcription; L, replication,recombination, and repair; M, cellwall/membrane/envelope biogenesis;N, cell motility; O, posttranslationalmodification, protein turnover,and chaperones; P, inorganic iontransport and metabolism; Q, secondarymetabolites biosynthesis, transport,and catabolism; R, general functionprediction only; S, function unknown;T, signal transduction mechanisms;U, intracellular trafficking, secretion, andvesicular transport; V, defense mecha-nisms; W, extracellular structures; X,mobilome, prophages, and transposons;Y, nuclear structure; Z, cytoskeleton;and NA, not assigned). Note that 99.7%of the aggregate length of genesclassified in the COG category Q in the“Ca. E. kahalalidefaciens” chromosome(red) correspond to NRPS pathwaysand occupy 20% of the genomecoding capacity. (B) Genetic organiza-tion of the 20 NRPS pathwaysin “Ca. E. kahalalidefaciens”(ordered by size), where eacharrow indicates a single gene.

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kahalalidefaciens2,345,455 bps

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Fig. 3. Structural and biosynthetic diversity of kahalalides fromBryopsis sp. (A) Molecular structures of nine kahalalides that can bebioinformatically linked to specific NRPS pathways in “Ca. E. kahalalide-faciens” and chemically detected in Bryopsis-2015. (B) Extracted ionchromatograms (EICs) for the mass/charge ratios (m/z) corresponding tothe (M + H)+ ions of the molecules in (A), as detected in the chemical extractof Bryopsis-2015. An asterisk indicates the peak corresponding to themolecule of interest. (C) Amino acid composition of the nine kahalalidesshown in (A), indicating the position and stereochemistry of each aminoacid on a linear scale. Kahalalide names are represented with two-letterabbreviations (e.g., kahalalide D = KD), and amino acids are denoted by their

canonical one-letter codes (A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His;I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr;V, Val; W, Trp; Y, Tyr) except for ornithine (O) and dehydrobutyrine (DB).FA, fatty acid. (D) Domain organization of “Ca. E. kahalalidefaciens”–encodedNRPS pathways that were bioinformatically linked to correspondingkahalalides in C (Cs, starting condensation domain; A, adenylation; T,thiolation; E, epimerization; C, condensation; and TE, thioesterase). Thenumber of modules in a given NRPS and the number and position ofepimerization domains encoded in it (black) match exactly what is observedin its linked product (see also substrate specificity analyses in figs. S3and S6 and detailed HR-MS/MS analyses in table S1 and figs. S1 and S7).

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all other kahalalides previously reported fromthis Bryopsis sp. First, consistent with the lipo-peptide nature of the kahalalides, the matchedNRPSs begin with a starting condensation do-main. Second, the number of modules in theNRPSs and the position of epimerization domainsin the modules match exactly the size and L- andD-amino acid positions in the correspondingmolecules. Third, the predicted substrate spec-ificity of adenylation domains in the NRPS(based on prediction algorithms and detailedphylogenetic analysis of the A domains) pre-dominantly match the amino acid compositionof the corresponding kahalalides (Fig. 3 andfigs. S4 and S6). Fourth, no other NRPS pathwaysrecovered from the metagenome are consistentwith the structure of any of the kahalalides(97 scaffolds). Finally, and most importantly, inevery case where we were able to bioinformat-ically match an NRPS in “Ca. E. kahalalidefaciens”to a corresponding kahalalide, we successfullyconfirmed the presence of the exact moleculein the chemical extract of Bryopsis-2015 usingHPLC–HR-MS/MS analysis (Fig. 3, fig. S7,and table S1). These results establish “Ca.E. kahalalidefaciens” as a symbiotic microbialfactory for at least nine complex molecules thatare abundant enough to be isolated from thealgal host. Of these nine molecules, at least onewas shown to be essential for the host’s chem-ical defense against predators.

An intracellular symbiont with areduced genome

Apart fromtheNRPS-coding regions, the remainingcoding capacity of the “Ca. E. kahalalidefaciens”genome is only 1.87 Mbps (average genome sizein the Flavobacteriaceae family is ~3.7 Mbps),indicating that this bacterium is undergoingevolutionary genome reduction. Next, we anno-tated the “Ca. E. kahalalidefaciens” genome usingthe Integrated Microbial Genome platform (21)and compared it to the closest free-living andgenome-sequenced bacterium:Mangrovimonassp. ST2L12 (88% DNA sequence identity for the16S rRNA gene) (22) (fig. S5). The genome of“Ca. E. kahalalidefaciens” encodes 873 protein-coding genes that can be functionally assigned

into a Cluster of Orthologous Groups (COG) cate-gory and 449 that can be classified into a KyotoEncyclopedia of Genes and Genomes (KEGG)pathway, whereas theMangrovimonas sp. ST2L12genome encodes 1868 and 801 such genes, respec-tively.Most enzymes and pathways forDNA repli-cation and protein synthesis, cofactor biosynthesis,and central and intermediary metabolism (gly-colysis, tricarboxylic acid cycle, pentose phosphatepathway, electron transport, and fatty acid syn-thesis and degradation) are present in both “Ca.E. kahalalidefaciens” and Mangrovimonas sp.ST2L12. However, some DNA repair genes andpathways (damaged nucleotide removal and baseexcision repair, nucleotidyltransferase involved inDNA repair, and RNA helicase), as well as genesinvolved in chemotaxis, detoxification, and adap-tations to atypical conditions, are mostly absentfrom “Ca. E. kahalalidefaciens,” a finding that iscommonly observed in obligate symbionts under-going genome reduction (23) (table S2).Most notably, no complete pathway to any of

the 20 proteinogenic amino acids can be iden-tified in the “Ca. E. kahalalidefaciens” genome,with most pathways missing all or the majorityof the genes. The only partially complete seriesare in the early steps of aromatic amino acidbiosynthesis leading to chorismate, but the down-stream enzymes for phenylalanine, tyrosine, andtryptophan biosynthesis are mostly absent (tableS2). Additionally, the “Ca. E. kahalalidefaciens”genome shows two features that are rarely foundin reduced genomes. First, its GC content hasnot been reduced: The average GC content of“Ca. E. kahalalidefaciens” is 52%, higher thanthat ofMangrovimonas sp. ST2-L12 (38%). Sec-ond, it contains an unusually high number (84) oftransposons, transposases, and transposable ele-ments.With this enrichment ofmobile elements,we wondered whether overt signs of horizontalgene transfer (HGT) can be detected in the ge-nome. We searched for signs of genome hetero-geneity bymultiple parametricmethods, includingGC content (24), k-mer frequencies (k = 2 or 3)(25), and dinucleotide bias (26). We detected atotal of seven genomic regions that stronglydeviate in their composition from the genomeaverage (fig. S8 and table S3). Six of these re-

gions range in size from 0.1 to 2.6 kbps andencode hypothetical proteins. A seventh regionof ~26 kbps (coordinates: 1821316 to 1847296)encodes several phage proteins, including cap-sid, portal, and tail sheath proteins, and likelyconstitutes a complete prophage. No other clearsigns of HGT were detected across the entiregenome, including the 20 NRPSs (fig. S8).Next,we testedwhether “Ca.E.kahalalidefaciens”

is capable of free living by attempting its culti-vation in various media and employing a sensi-tive, high-throughput sequencing–based methodfor its detection (see materials and methods andfig. S9). These efforts, however, were not suc-cessful, suggesting that “Ca. E. kahalalidefaciens”is an obligate symbiont. Indeed, most genome-reduced, obligate symbionts live intracellularlyin their hosts (23). To determine whether “Ca.E. kahalalidefaciens” is an intracellular or extra-cellular symbiont of Bryopsis sp., we performedfluorescence in situ hybridization (FISH) on fixedBryopsis-2015 specimens using fluorescent probesthat target regions of the 16S rRNA. Two sets ofprobes were used: a combination of universal eu-bacterial probes EUB338 I, II, and III (27, 28) andone that is specific for “Ca. E. kahalalidefaciens”(JZP2; seematerials andmethods). Both general andspecific probes localized “Ca. E. kahalalidefaciens”to the cellular compartment of Bryopsis, wheremost detected signals were from dispersed cellswithin the algal cytosol (Fig. 4). Notably, wefound that a partial 16S rRNA gene sequence(with 100% DNA sequence identity to that of“Ca.E. kahalalidefaciens”) was previously clonedfrom a Brazilian sample of Bryopsis sp. (fig. S5)(29). This previous study suggested that the bac-terium harboring this sequence is also intra-cellular and widespread in Bryposis sp. livingin warm-temperate and tropical environments(30). Finally, we searched a recently publisheddataset composed of 16S rRNA gene ampliconsequences from 1194 sponges, 37 sediments, and195 seawater samples for sequences that match“Ca. E. kahalalidefaciens” (31). At a cutoff of 97%DNA sequence identity, nomatches were found,further supporting the specific, obligate, andintracellular symbiotic relationship between“Ca. E. kahalalidefaciens” and Bryopsis sp.

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Fig. 4. Localization of “Ca. E. kahalalidefaciens” in Bryopsis sp. using FISH. Epifluorescent micrographs of a Bryopsis sp. section hybridized with(A) universal eubacterial probes EUB338 I to III (labeled with Cy3, red) and (B) “Ca. E. kahalalidefaciens”–specific probe JZP2 (labeled with 6-FAM,green). (C) Algal cell wall of the same section viewed under a 4′,6-diamidino-2-phenylindole (DAPI) channel using calcofluor counterstaining.(D) Composite of images (A) to (C), showing the colocalization of the red and green signals for “Ca. E. kahalalidefaciens” but not for other bacteria.Arrows indicate bacteria: cEK, “Ca. E. kahalalidefaciens”; OB, other bacteria.

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Given that there is no clear nutritional ad-vantage provided by “Ca. E. kahalalidefaciens”and that 20% of the bacterial genome is dedi-cated to the biosynthesis of complex small mole-cules, we assume that the most important aspectof this relationship is chemical defense in exchangefor a tolerant intracellular environment in whichto live. Notably, “Ca. E. kahalalidefaciens” is de-fective in synthesizing all 16 amino acids thatare incorporated as substrates during the bio-synthesis of the kahalalides (Fig. 3). These sub-strates are provided by the autotrophic Bryopsissp. host, although other intracellular bacteria mayalso contribute. These findings establish an un-usual collaborative model for the biosynthesisof the kahalalides, where the host provides theinitial substrates and the symbiont provides thebiochemical machinery to transform these sim-ple substrates into complex, biologically activemolecules that may otherwise be unattainablein algal biochemistry.

A tripartite symbiosis

Through kleptoplasty, E. rufescens sequestersand maintains intact and functioning chloro-plasts from the cells of its Bryopsis sp. diet. Canthe mollusk likewise “steal” intact intracellularbacterial symbionts of Bryopsis sp. (e.g., “Ca.E. kahalalidefaciens”)? To answer this question,we collected several specimens of E. rufescens,confirmed that they contain KF, and then useddeep 16S rRNA gene amplicon sequencing fromwhole-animal metagenomic DNA of three indi-viduals (an average of 57,000 reads per sample)to search for “Ca. E. kahalalidefaciens.”The E. rufescensmicrobiome is dominated by

sequencesmatching the algal chloroplast (47.0%,on average), Mollicutes (29.0%), and Gamma-proteobacteria (12.5%) (fig. S10), in agreementwith a previous study (32). Despite the sequenc-ing depth performed in our study, we barelydetected 16S rRNA gene sequences that match“Ca. E. kahalalidefaciens” in E. rufescens (anaverage of 0.06% of the obtained reads). Addi-tional sequencing of 16S rRNA gene ampliconsfrom sections of two coarsely dissectedE. rufescensindividuals (an average of 81,000 reads persection) confirmed that there was no specificsymbiosis organ in the mollusk and that “Ca.E. kahalalidefaciens” cells appear to be fullydigested upon ingestion (fig. S10). Finally, thoughwe could clearly observe signals for other bacte-ria using general eubacterial probes, negative FISHexperiments on severalE. rufescens sections usinga specific probe for “Ca. E. kahalalidefaciens”corroborated our sequencing results (fig. S11).Taken together, our results establish chemicalbut not symbiont sequestration as a likely meansof KF acquisition, although the mechanistic de-tails of this step are unknown.

A model for the evolution ofdefense chemicals

Next, we asked what molecular mechanismsgenerate the observed diversity in kahalalidestructures. Because we found no evidence ofHGT-mediated acquisition of NPRS pathways

in the “Ca. E. kahalalidefaciens” genome (fig. S8),we looked for signs of increased genome plas-ticity (33, 34) and intragenome genetic exchange.Briefly, we segmented the genome into 150-bpfragments, produced a whole-genome identitydot plot by comparing all fragments, and searchedfor genomic loci that contain a high density ofmatched fragment pairs (>60% sequence iden-tity). On average, NRPS loci showed a 50-foldhigher density of matched fragments than therest of the genome (Fig. 5A; see also materialsand methods and fig. S12), in a largely indis-criminate (matched fragments within a singleNRPS pathway are neither more frequent normore similar than those between pathways)(fig. S13) and pervasive (matched fragmentscover >98.8% of the combined NRPS sequences)manner. A notable outlier in this analysis wasNRPS-8, where matched fragments betweenNRPS-8 and other pathways showed lowerdensity and percent identity than between anyother pairs of NRPSs (Fig. 5A and figs. S12and S13).Consecutivematched fragments between pairs

of NRPSs form visually distinct stripes on iden-tity dot plots, which represent alignments thatstart and end with abrupt changes in DNA se-quence identity as a result of possible geneticexchange events (Fig. 5B and fig. S12). We iden-tified more than 100 such events: Three repre-sent full pathways and can therefore be explainedby typical duplication and divergence events (onebetween NRPS-10 and NRPS-13; two betweenNRPS-3, NRPS-12, and NRPS-20), whereas theremaining ones represent only parts of thepathways and are best explained by separaterecombination events (Fig. 5C and fig. S14). Inmost cases, lineage trees for different alignedsequences that appear in the same set of NRPSsdo not follow the same path (Fig. 5D), indicat-ing that frequent shuffling substantially dis-rupts the trajectories of pathway duplication.Finally, 9 of the 20 NRPSs contain or occurwithin a 5-kbp window of an annotated trans-posase or transposon (fig. S15), suggesting thattransposition is at least partially responsible forthe observed plasticity, as seen in other systems(33, 34). These findings motivated us to proposea new model for the diversifying evolution ofNRPSs in the “Ca.E. kahalalidefaciens” genome,where new pathways arise through duplicationand divergence events that are concurrently ac-companied by a high frequency of interpathwayrecombination, resulting in the observed diver-sity and an intertwined evolutionary history.Why would a bacterial symbiont produce so

much chemical diversity? In other words, arethese molecules equally important in establish-ing a sustainable symbiotic relationship, espe-cially given thatmaintaining and expressing theirlarge biosynthetic pathways is a major metaboliccost? We reasoned that nonessential pathwaysshould decay and eventually get lost, whereasessential ones should be intact and maintained.We looked for signs of genomic decay in the20 NRPS pathways. Indeed, NRPS-4 and NRPS-16 show truncated domains and modules, and

NRPS-2, NRPS-7, and NRPS-9 contain domainsthat have been interrupted by transposases(Fig. 2B). The remaining NRPS pathways appearto be pristine. We then compared the expressionlevels of the NRPS pathways under native con-ditions, reasoning that essential pathways wouldbe highly expressed, whereas decaying ones wouldbe expressed at a lower level or not expressed atall. Metatranscriptomic sequencing data fromBryopsis-2015 (40 million paired-end reads,150 bps; 41 million single-end reads, 100 bps)were mapped to the “Ca. E. kahalalidefaciens”genome (see materials and methods), resultingin the successful alignment of 2.2 million reads(mostly to possible mRNAs) (Fig. 6A). We thenranked all genes according to their expressionlevel (data S1), which produced a trimodal dis-tribution: About 50% of the genes are almostsilent or have a very low level of expression, 45%are at median level, and 5% are among the mosthighly expressed (Fig. 6B). Comparison of theexpression level of the NRPS genes showed asimilar distribution. Genes from NRPS-1, -2, -4,-6, -9, -16, and -17 (NRPS-2, -4, -9, and -16 werepredicted to be decaying pathways) had thelowest expression levels, and genes fromNRPS-8had the highest. NRPS-8 genes were among themost highly expressed genes in the entire ge-nome, along with cell division and transcrip-tion genes. NRPS-8 alone accounted for morethan 12% of the transcriptional activity of “Ca.E. kahalalidefaciens,” and in total, all of theNRPS pathways accounted for 26% (Fig. 6A).These results establish NRPS-8 (encoding themost toxic kahalalide, KF) as a potentially es-sential pathway, and NRPS-2, -9, and -16 as po-tentially on their way to being lost.We thus propose a model for NRPS path-

way generation, evolution, and selection in “Ca.E. kahalalidefaciens” (supplementary text). Inour model, new pathways are created from anancestral one through duplication and diver-gence, followed by extensive and continuousinterpathway genetic exchange to generatemore diversity. Newly generated pathways arethen selected for or against, depending on un-known selection pressures, leading to theirmaintenance or loss, followed by a continuousrepetition of the same cycle (fig. S16).

Discussion

KF is themost toxic of all known kahalalides andis likely the main contributor to Bryopsis sp. andE. rufescens chemical defense against predators(7). KF andmore than 15 related kahalalides havebeen reported from this predator–prey system,yet the original source of this library ofmoleculeshas been unknown. In this study, we used meta-genomics, metatranscriptomics, chemical analy-sis, microscopy, and evolutionary genomics touncover the molecular details of kahalalide pro-duction by an elusive third partner, an intra-cellular bacterial symbiont of Bryopsis sp.Chemically defendedmollusks are widespread

in the marine environment, but the source oftheir defense chemicals varies (5). Although chem-ical sequestration from diet has been proposed

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in many cases (35), de novo biosynthesis hasalso been demonstrated through isotope label-ing experiments (36). Here, we provide evidencethat defense chemicals in E. rufescens do not

originate in the algal diet itself but in intra-cellular bacterial symbionts within the alga. Asimilar indirect acquisition mechanism may betrue for other chemicals found inmollusks, such

as those acquired from dietary sponges andtunicates. Most work on symbioses in marinealgae has focused on extracellular symbionts, asexemplified by the symbiosis between Emiliania

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Fig. 5. Intensive genetic exchange in “Ca. E. kahalalidefaciens” NRPSpathways. (A) Pairs of sequence fragments sharing more than 60% identityacross the whole “Ca. E. kahalalidefaciens” genome, shown on a linear scale onboth the x and y axes. Diagonal matches were removed, and color indicatespercent identity. Positions of the 20NRPS pathways are indicated on the axes,matching the color code below. (B) Examples of aligned sequences betweenNRPS pathways. Note the abrupt rise and fall in sequence identity. Colorsin pathways indicate different domains, matching the color code below.Encoded amino acids are indicated by their single-letter abbreviations, andunderlined letters indicate D-amino acids. (C) Circular representation of“Ca.E. kahalalidefaciens”genome recombination events (coordinate one of thegenome is indicated by a solid black line), in which NRPS pathways areshown at their respective positions around the genome and connecting colored

lines indicate identified pairwise recombination events. Lines follow a yellow-to-blue color code that represents the percent identity between pairs of alignedsequences, and their thickness positively correlates with the length of thesequence. Sequences shared with NRPS-8 and NRPS-9 have a lower percentidentity than ones shared with other pathways. Black lines indicate the threeidentified duplication events. (D) Phylogenetic trees for three groups of alignedsequences (red, green, and blue) that together cover the entire NRPS-15.Although the three aligned sequences are found in NRPS-15, they do not followthe same phylogenetic lineage and are likely a result of three independentrecombination events. NRPS-15 is shown as a black rectangle, and NRPSpathways that share one ormore of the three groups of aligned sequenceswithit are shown as gray rectangles.The scale bar at top right indicates 10%sequence divergence.

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huxleyi and Phaeobacter inhibens, in whichP. inhibens influences the biology of the hostthrough alternating symbiotic and pathogeniccycles (37), and that between Ulva sp. andseveral bacteria, in which the symbionts triggerproper host growth and development (38). Here,we describe the functional importance of intra-cellular symbionts of marine macroalgae for theecology of their host, which has been previouslyoverlooked. We also establish macroalgae as anadditional group of marine organisms in whichbacterial symbionts produce complex naturalproducts discovered in the host, as has beenpreviously observed inmarine tunicates, sponges,bryozoans, and crustaceans (4).The diversifying evolution of NRPS path-

ways in the “Ca. E. kahalalidefaciens” genomeis particularly noteworthy because it reveals astrategy for chemical innovation in bacteria ina HGT-independent manner. The closest exam-ples are two cases reported in bacterial symbiontsof marine ascidians: one in which the samepolyketide synthase pathway has duplicatedmultiple times in the same genome, presumablyresulting in higher expression levels (39), andanother in which a cyanobactin pathway has dup-licated once and accumulated strategic pointmutations in its precursor peptide to producenovel molecules (40, 41). The number of bio-synthetic pathways (20 NRPSs) and resulting mol-ecules discovered in “Ca. E. kahalalidefaciens”is substantial, especially given its reduced ge-nome and strict intracellular lifestyle, and ap-proaches the diversity encoded in the genomeof Entotheonella sp., an extracellular symbiontof marine sponges and a talented producer ofnatural products (42, 43).

We do not yet understand the nature of the se-lective pressures on the “Ca. E. kahalalidefaciens”genome. Assuming that all kahalalides have rolesin chemical defense, we propose either that theycontribute to a synergistic cocktail, where mole-cules function additively on diverse predators orpathogens and the selection acts to maintaindiversity, or that eachmolecule has a specific roleand is selected for independently. There is somesupport for the latter hypothesis in that KF hasbeen shown to have a strong antipredatory ac-tivity (7), and the rest of the kahalalides have lowto no cytotoxicity or antimicrobial activity andtheir natural role has not yet been determined(9). Furthermore, levels of different kahalalidesproduced in the same system vary considerablybetween times of collection (17), suggesting thatthey are selected for individually and not globally.We also do not understand how E. rufescenssequesters, concentrates, and resists the cyto-toxicity of the kahalalides. This is complicatedby the fact that their molecular target is stillunknown, even for KF, which has been evaluatedas a promising anticancer agent in several hu-man clinical trials (44). We show that chemicaland not symbiont sequestration is responsiblefor kahalalide acquisition in E. rufescens, butexactly how and where this occurs await furtherinvestigation. These outstanding questions arenot specific to E. rufescens and the kahalalidesbut are true for almost all marine mollusks thatsequester toxins from their diet.

Materials and methods summary

An expanded description of materials andmethods can be found in the supplementarymaterials.

Bryopsis sp. and E. rufescens collectionand processingBryopsis sp. and E. rufescens samples were col-lected from Black Point Bay, Honolulu, Hawaii(N21°15′34″; W157°47′24″). Small portions ofBryopsis sp. and individual E. rufescens speci-menswere preserved frozen for chemical analysis,preserved in RNAlater (ThermoFisher Scientific,USA) for DNA and RNA analysis, or fixed inparaformaldehyde for FISH analysis.

Metagenomic DNA and RNA extractionand sequencing

Total metagenomic DNA used for both Illuminaand Pac Bio sequencing was extracted from ly-ophilized Bryopsis sp. and E. rufescens usingthe Mo Bio Power Biofilm DNA isolation kit(Mo Bio Laboratories, USA; now Qiagen, USA).Total RNA was extracted from RNAlater-preservedBryopsis sp. using MasterPure Complete DNAand RNA Purification Kit (Epicentre, USA; nowIllumina, USA). Illumina sequencing was per-formed on an Illumina HiSeq 2500 sequencer(Illumina, USA), and Pac Bio sequencing wasperformed on a PacBio RS II instrument (PacificBiosciences, USA).

Fluorescence in situhybridization (FISH)

Fixed specimens ofBryopsis sp. were embeddedin LRWhite resin (ElectronMicroscopy Sciences,USA), cut into 2-mm-thick slices, hybridized witheither universal eubacterial probes or a “Ca.E. kahalalidefaciens”–specific probe, counter-stained using Calcofluor White Stain (SigmaAldrich, USA), and imaged on a Leica SP8 con-focal microscope.

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Fig. 6. Metatranscriptomic analysis of “Ca. E. kahalalidefaciens.”(A) Genome-wide transcriptional activity of “Ca. E. kahalalidefaciens”using metatranscriptomic data collected from Bryopsis-2015. Black barsindicate total counts for each mapped position and averaged per 5 kbps ofthe genome. The inset bar graph indicates the ranked contribution ofeach NRPS pathway as a percentage of the total genome transcriptional

activity. (B) “Ca. E. kahalalidefaciens” genes (x axis, shown in percentageof the total number of genes in the genome) plotted against theircumulative expression (y axis, shown in percentage of the total expressionof all genes in the genome). Note the trimodal distribution of genesaccording to their expression and that genes from the NRPS pathwaysappear in all three modes.

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Bacterial cultivation and screening for“Ca. E. kahalalidefaciens”

Freshly collected Bryopsis sp. (5 g) was homo-genized in 9 ml of sterile 1X Artificial Sea Water(Instant Ocean Sea Salts, 35 g/liter, InstantOcean, USA) using a mortar and pestle, andthe algal homogenate was serially diluted in a10-fold series. 100-ml aliquots of each dilutionwere plated on eight different media in tripli-cate, and plates were incubated at room temper-ature for 1 to 2 weeks. Colonies were scrapedfrom one plate of each of the eight media, andDNAwas extracted from themixture and screenedfor the presence of “Ca. E. kahalalidefaciens”using high-throughput 16S rRNA gene ampli-con sequencing.

Chemical extraction and analysis ofBryopsis-2015

Lyophilized Bryopsis sp. was extracted withmethanol and analyzed by HPLC–HR-MS/MSusing an Agilent 6500 Series Q-TOF LC/MS sys-tem (Agilent, USA).

Evolutionary analysis of the “Ca.E. kahalalide” genome and the 20 NRPSs

The “Ca. E. kahalalidefaciens” genome was seg-mented into fragments of 150 bps in length,with a 30-bp sliding window. Each fragmentwas aligned to all other fragments to obtaintheir percent identity; pairs of fragments withidentities higher than 60% were recorded as“matched.” Consecutive matched fragments be-tween two genomic regions form stripes with aslope of 1 and varying intercepts on identity dotplots. These stripes were then used to infer ge-netic exchange events throughout the genome.

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ACKNOWLEDGMENTS

We are indebted to J. Davidson and the late Ruth Gates at HIMBfor laboratory space and assistance during the field work conductedin this study. We thank G. Laevsky, the Molecular BiologyConfocal Microscopy Facility (a Nikon Center of Excellence), J. Yan,and B. Bassler for assistance with confocal microscopy; W. Wangand the Lewis Sigler Institute sequencing core facility forassistance with high-throughput Illumina and 16S rRNA geneamplicon sequencing; L. Tallon, L. Sadzewicz, and the Institute forGenome Sciences, Genomics Resource Center, University ofMaryland, for Pac Bio sequencing; M. Cahn and J. Lopez forassistance with metagenomic data analysis; M. T. Hamann,W. L. Cheung-Lee, and A. J. Link for valuable scientific insightsabout the project; S. Chatterjee for general assistance; and the restof the Donia lab for useful discussions. Funding: Funding for thisproject has been provided by Princeton University, M.S.D. is fundedby an NIH Director’s New Innovator Award (ID 1DP2AI124441), andZ.L. is supported by Princeton Center for Theoretical Science andCenter for the Physics of Biological Function, the National ScienceFoundation Physics Frontier Center grant through the Center forthe Physics of Biological Function (PHY-1734030), and an NSF

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grant (PHY-1607612). Author contributions: M.S.D., J.Z., Z.L., andR.T.H. designed the study. J.Z., Z.L., M.D.T., J.D., and M.S.D.performed experiments and analyzed the data. M.S.D., J.Z., Z.L.,and M.D.T. wrote the manuscript. J.D. and R.T.H. edited themanuscript. Competing interests: The authors declare nocompeting interests. Data and materials availability: All data areavailable in the main text or the supplementary materials. The“Ca. E. kahalalidefaciens” genome has been deposited to the

Integrated Microbial Genomes (Joint Genome Institute, U.S.Department of Energy) public repository under IMG submissionID 115642.

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science.sciencemag.org/content/364/6445/eaaw6732/suppl/DC1Materials and Methods

Supplementary TextFigs. S1 to S16Tables S1 to S3References (45–58)Data S1

15 January 2019; accepted 7 May 201910.1126/science.aaw6732

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A microbial factory for defensive kahalalides in a tripartite marine symbiosisJindong Zan, Zhiyuan Li, Ma. Diarey Tianero, Jeanette Davis, Russell T. Hill and Mohamed S. Donia

DOI: 10.1126/science.aaw6732 (6445), eaaw6732.364Science 

, this issue p. eaaw6732; see also p. 1034Sciencetoxins but, to protect itself from being eaten by fish, grazes on the alga to accumulate kahalalide.authors elucidated the pathways for generating this chemical diversity. It seems that the sea slug not only tolerates thea very reduced genome was discovered to be a factory for the nonribosomal assembly of a family of kahalalides. The

withinvolved in toxin production (see the Perspective by Mascuch and Kubanek). Within the alga, a species of bacterium wondered if a third party waset al.predators using peptide toxins decorated with fatty acids, called kahalalides. Zan

sp. The alga defends itself fromBryopsis grazes on an alga called Elysia rufescensThe Hawaiian sea slug A little help from a friend

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REFERENCES

http://science.sciencemag.org/content/364/6445/eaaw6732#BIBLThis article cites 57 articles, 7 of which you can access for free

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