Synergistic Allelochemicals from a Freshwater Cyanobacterium

Synergistic allelochemicals from a freshwater cyanobacterium

Pedro N. Leãoa,b,1, Alban R. Pereirab,1, Wei-Ting Liuc, Julio Ngd, Pavel A. Pevznerd, Pieter C. Dorresteinb,c,e, Gabriele M. Königf, Vitor M. Vasconcelosa,g,2, and William H. Gerwickb,e,2

aCIIMAR/CIMAR-LA, Center for Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal; bScripps Institution of Oceanography, cDepartment of Chemistry and Biochemistry, dDepartment of Computer Science and Engineering, and eSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093-0636; fInstitute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany; and gDepartment of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved May 17, 2010 (received for review December 12, 2009)

The ability of cyanobacteria to produce complex secondary meta- very limited number of allelochemicals have been identified and bolites with potent biological activities has gathered considerable mainly from freshwater cyanobacteria (20). These include cyano- attention due to their potential therapeutic and agrochemical ap- bacterin, a chlorinated γ-lactone produced by Scytonema hofman- plications. However, the precise physiological or ecological roles ni that inhibits other cyanobacteria and green microalgae (21); played by a majority of these metabolites have remained elusive. fischerellin A, an enediyne-containing photosystem II inhibitor Several studies have shown that cyanobacteria are able to inter- produced by Fischerella muscicula (22); the hapalindoles, small fere with other organisms in their communities through the release metabolites that have been isolated from Hapalosiphon and of compounds into the surrounding medium, a phenomenon usual- Fischerella spp. that inhibit several microorganisms (23, 24); ly referred to as allelopathy. Exudates from the freshwater cyano- and the nostocyclamides, relatively small cyclic peptides from bacterium Oscillatoria sp. had previously been shown to inhibit the Nostoc sp. that inhibit cyanobacteria and microalgae (25, 26). green microalga Chlorella vulgaris. In this study, we observed that Different ecological roles have been attributed to the production maximal allelopathic activity is highest in early growth stages of the of allelochemicals by cyanobacteria, including phytoplankton cyanobacterium, and this provided sufficient material for isolation succession, bloom formation, resource and interference competi- CHEMISTRY and chemical characterization of active compounds that inhibited tion (27), and invasive fitness (28). In order to understand the the growth of C. vulgaris. Using a bioassay-guided approach, we ecological significance of allelopathy in cyanobacteria, several isolated and structurally characterized these metabolites as cyclic studies have characterized environmental factors that may mod- peptides containing several unusually modified amino acids that ulate allelopathic events. The presence of competitors (29–31) are found both in the cells and in the spent media of Oscillatoria and coexisting heterotrophic bacteria that degrade allelochemical sp. cultures. Strikingly, only the mixture of the two most abundant substances (32) have been identified among biotic factors. Light

metabolites in the cells was active toward C. vulgaris. Synergism intensity (33), temperature (34), nutrient levels, and pH (35) have ECOLOGY was also observed in a lung cancer cell cytotoxicity assay. The binary been shown to control allelochemical production in some species mixture inhibited other phytoplanktonic organisms, supporting of cyanobacteria. a natural function of this synergistic mixture of metabolites as We had previously reported (36) the inhibitory effect of exu- allelochemicals. dates from the mat-forming cyanobacterium Oscillatoria sp. strain LEGE 05292 (OSC) on the microalga C. vulgaris. Here, we eval- allelopathy ∣ chemical ecology ∣ Oscillatoria ∣ cyclic peptides ∣ synergism uated the influence of abiotic factors and culture conditions on this allelopathic interaction and found that the main factor con- yanobacteria are a prolific source of nearly 800 diverse bioac- trolling activity was the growth stage of the OSC cultures. Using Ctive secondary metabolites, originating mainly from nonribo- bioassay-guided fractionation of the OSC biomass, we isolated one somal peptide synthetase (NRPS) or mixed polyketide synthase of the compounds responsible for the inhibitory activity (portoa- (PKS)–NRPS biosynthesis (1, 2). Efforts to isolate and character- mide A, 1) in sufficient quantity for structural characterization by ize cyanobacterial metabolites have usually been motivated from nuclear magnetic resonance (NMR). Confirmation of the struc- a desire to describe either their natural toxicity toward animals in ture of 1 and its homolog (portoamide B, 2) was achieved by natural settings (e.g., ref. 3) or promising activities from in vitro state-of-the-art mass spectrometric (MS) sequencing methodolo- biomedical screening programs (e.g., ref. 4, 5). However, in general gies (37). A chemoenzymatic approach together with MS data the ecological role played by the majority of these metabolites is allowed us to deduce the structures of the related compounds por- not well known (6, 7). Functions established to date for cyanobac- toamides C and D (3 and 4, respectively). The four metabolites terial secondary metabolites include nitrogen storage (8), UV pro- were found to be present both in the cells and in the culture med- tection (9), metal chelation (10), defense against predation (11), ium of actively growing OSC, thus supporting their role as allelo- and quorum sensing (12). Allelopathy refers to the chemically mediated interaction between plants or microorganisms (13). These interactions are Author contributions: P.N.L., A.R.P., W.-T.L., J.N., P.A.P., P.C.D., V.M.V., and W.H.G. designed research; P.N.L., A.R.P., W.-T.L., J.N., P.A.P., and P.C.D. performed research; P.N.L., A.R.P., characterized by the release of allelopathic compounds (allelo- W.-T.L., J.N., P.A.P., P.C.D., V.M.V., and W.H.G. analyzed data; G.M.K. contributed new chemicals) into the surrounding medium, eliciting either a posi- reagents/analytic tools; and P.N.L., A.R.P., V.M.V., and W.H.G. wrote the paper. tive or deleterious response in a target organism (13). In aquatic The authors declare no conflict of interest. ecosystems, allelopathy is regarded as an important process This article is a PNAS Direct Submission. – influencing the shaping of microbial communities (14 16). Toxic Data deposition: The DNA sequence reported in this paper was deposited in the GenBank properties have been attributed to cyanobacteria over the last database (accession no. GU085101) 130 yr (e.g., ref. 17), and the allelopathic potential of these organ- 1P.N.L. and A.R.P. contributed equally to this work. isms was described through field-derived observations in the 2To whom correspondence may be addressed. E-mail: [email protected] and wgerwick@ 1970s (18, 19). Since then, several genera of cyanobacteria have ucsd.edu. been implicated in allelopathic phenomena, with targets ranging This article contains supporting information online at www.pnas.org/lookup/suppl/ from other cyanobacteria to higher plants; unfortunately, only a doi:10.1073/pnas.0914343107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914343107 PNAS Early Edition ∣ 1of6 Downloaded by guest on October 2, 2021 chemicals. Structurally, they are cyclic peptides containing several unusually modified amino acids. Strikingly, we found that only the mixture of 1 and 2 was effective in the inhibition of C. vulgaris −1 growth (IC50 ¼ 12.8 μg·mL ). This mixture inhibited other phytoplanktonic organisms, and synergism was also observed in an in vitro cancer cell cytotoxicity assay. Results Identification of the Producing Strain (OSC). We identified this strain as Oscillatoria sp. in our previous report (36). The genus Oscilla- toria was among the closest matches in the databases regarding the 16S rRNA gene sequence data (see SI Text for details).

Influence of Biotic and Abiotic Factors on Allelopathic Activity. We analyzed the influence of different light, temperature, and nutrient stress conditions on the ability of filtrates from OSC cultures Fig. 2. Major secondary metabolites isolated from OSC biomass or media. to inhibit the growth of C. vulgaris. Low-light, high-light, low- Numbering corresponds to portoamide A (1). temperature, or nutrient limitation did not increase activity when compared to control conditions (Fig. S1). However, when cell tively large metabolite structure. Combining this information with densities and culture stage conditions were evaluated, a peak in NMR data (Table S1), it was possible to derive a molecular for- the inhibitory activity of the filtrates (∼11–27% of growth relative mula of C74H109N13O22 (calculated for C74H109N13O22Na, to control) was observed between days 10 and 15 for all cultures, 1554.7702), exhibiting 27 degrees of unsaturation. corresponding to the beginning of the exponential growth phase Extensive analysis by 2D NMR, including heteronuclear single as inferred from nitrate consumption. Filtrates obtained even at quantum coherence (HSQC), heteronuclear multiple bond very early time points displayed considerable activity (e.g. at correlation (HMBC) (Fig. S4), COSY, and NOESY (Fig. S5), re- 3 d, the filtrate had between 28–89% of growth relative to controls) vealed the presence of nine standard and two uncommon amino (Fig. 1). acids, together with an unusual β-amino acid (Fig. 3A). As shown Bioassay-Guided Isolation and Structural Elucidation of Portoamides A in Fig. 3A, a combination of COSY and HMBC data led to an unambiguous assembly of spin systems for the following standard (1) and B (2). After harvesting and filtration (see SI Text), the lyo- 1 ∕ amino acid residues: Pro (×3), Thr, Ser, Ile, Gly, Gln, and Tyr. H philized biomass was repeatedly extracted with CH2Cl2 MeOH 13 (2∶1) and fractionated by silica gel chromatography. The most and C chemical shifts for these building blocks were in agree- polar fraction (100% methanol) showed high growth inhibitory ment with literature values (38, 39). Interestingly, Tyrwas found to N activity against C. vulgaris and was thus subjected to reverse- be both methylated and acetylated at its terminus, as revealed by δ phase HPLC to afford cyclic dodecapeptide 1 and its closely re- HMBC correlations from methyl singlets H72 ( 2.77) and H74 δ δ α lated minor analogue 2 (Fig. 2). The smaller metabolites 3 and 4 ( 1.82) to carbonyl C73 ( 170.4) and -methine carbon C63 were also present in this fraction (Fig. S2A) but in insufficient (δ57.6). To our knowledge, this is a previously undescribed com- quantity for NMR structural analysis. bination of appendages detected for Tyr in a cyanobacterial- 1 The H-NMR spectrum of 1 in DMSO-d6 (Table S1 and derived natural product. Fig. S3A) displayed a large number of very broad resonances, sug- The uncommon amino acid, O-methylhomotyrosine (MeHty), gesting both the presence of multiple conformers in solution and was assembled in analogy to the Tyr residue above. However, an a relatively large compound. Differentiated regions for NH pro- additional methylene (H28, δ1.88) was detected as part of the tons (δ8.45-7.10), α-methine multiplets (δ5.10-4.10), O-methyl, spin system comprised by α-methine H7 (δ4.29) and the benzylic and N-methyl singlets (δ3.70 and δ2.77 respectively), as well as diastereomeric methylene H9 (δa2.47 and δb2.58). The unique alkyl chain methyl doublet and triplets (δ0.89-0.80), revealed O-methyl singlet (H71, δ3.70) in 1 displayed an HMBC correla- the peptidic nature of compound 1. Furthermore, numerous re- tion with quaternary carbon C13 (δ157.4) in the 1,4-disubstituted sonances between δ7.18-6.50 suggested the occurrence of aromatic amino acid residues. High resolution electrospray ionization mass spectrometry (HRESIMS) analysis yielded an ½M þ Naþ parent ion at m∕z 1554.7695, accompanied by a more intense ½M þ 2Na2þ peak at m∕z 788.8793, confirming a rela-

Fig. 1. Allelopathic activity of filtrates from OSC grown at different cell Fig. 3. Selected HMBC, COSY, and NOE correlations for 1 indicating: (A)All densities. (A) Growth of C. vulgaris in the OSC filtrates retrieved at different the amino acid residues identified. (B) Interresidue connectivity. (N or O:sus- growth stages. (B) Growth profile of the OSC cultures, as inferred from pected heteroatom attached to the carbonyl group included in the following nitrate removal from the medium. fragment).

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0914343107 Leão et al. Downloaded by guest on October 2, 2021 aromatic ring of O-MeHty. Dehydrobutyrine (Dhb) in turn was elucidated from HMBC correlations between the highly diagnos- tic vinylic proton H22 (δ5.56) and the two sp2 carbons C21 (δ130.3) and C20 (δ164.7), forming part of an α,β-unsaturated amide moiety. The remaining substructure comprising 1 was found to be the β-amino acid 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Aound), given the striking similitude between our 1H and 13C NMR structural data (see SI Text) and that reported for the same fragment in schizotrin A (40) and pahayokolides A and B (38). A summation of the above partial structures (Fig. 3A) accounted for all atoms defined in the molecular formula and possessed 26 of the required 27 degrees of unsaturation, thus indicating that compound 1 contained an additional ring structure. Next, we sought to determine the connectivity of the above 12 residues. The lack of well-defined interresidue correlations from the HMBC spectrum (possibly due to line broadening) led us to approach this task using NOE data, which led us to the final amino acid sequence Gly-Gln-Aound-Pro1-O-MeHty-Thr-Dhb- Ser-Ile-Pro2-Pro3. (Fig. 3B and SI Text). The remaining N-acetyl-N-methyltyrosine (N-Ac-N-MeTyr) appendage was connected via an ester linkage to C54 (δ69.8), on the basis of the deshielded chemical shift value exhibited by its attached proton H54 (δ5.05). This proposed amino acid sequence from NOE and chemical shift data was validated via comparative dereplication (37), a novel mass-spectrometry-based computational approach for sequencing cyclic peptides given known structurally related compounds. Using this web tool, the experimental spectrum of compound 1 was CHEMISTRY searched against a database of nonribosomal peptides that had been manually supplemented with the sequences of tychonamide A and B. The algorithm takes the raw mass spectral information and identifies the closest match in the database. The top comparative dereplication matches were sequences derived from tychonamide A, confirming their structural similarity. No other sequence

in the database had significant matches to the MS spectrum of Fig. 4. Connectivity of amino acid residues in 1, based on selected MALDI- ECOLOGY compound 1. Once a good match was found, the observed frag- TOF-MS2 fragmentations. The cyclic arrangement of the selected fragments is ment ions were compared, and mass shifts of differing ions as well shown (top), together with their respective identification and m∕z values as similar mass differences compared to the parent ion allowed (bottom). rapid identification of identical versus differing portions of these two molecules. In doing so, the algorithm identified that the rence of LðSÞ-Pro and N-Me-LðSÞ-Tyr (see SI Text). With this, 11 46.022 Da mass difference between tychonamide A and compound of the 16 stereogenic centers in the planar structure of 1 were 1 was located on the Aound unit (see Fig. S6 A and B). An algo- assigned. The geometry of the double bond in Dhb was found rithmic description of this procedure is detailed in Ng et al. (37). to be E on the basis of strong NOE correlations between methyl δ δ 7 76 δ Finally, with the location of the modified residue in hand, all of the H19 ( 1.09) and C17-NH ( H . ) with methyl H23 ( 1.86) in the ions in a MS2 spectrum were annotated with our annotation script double bond. 1 MS-cyclic peptide annotation (MS-CPA)program (41) (Fig. S6C). Portoamide B (2) showed a H NMR (Fig. S3B) very similar to Fig. 4 displays a small fraction of representative fragments de- that obtained for portoamide A (1) with the exception of an rived from MALDI-TOF MS2 measurements on portoamide A absent aromatic methoxy singlet at δ3.70 (O-MeHty residue). (1). Taken together, fragments A–M supported the linear amino The absence of a mass equivalent to a -OCH2- fragment was con- þ acid sequence Gly-Pro3-Pro2-Ile-Ser-Dhb-Thr-O-MeHty-Pro1- firmed by HRESIMS, which displayed an ½M þ Na parent ion Aound-Gln, confirming the connectivity derived from NOE data. at m∕z 1524.7541 and was accompanied by an intense 2þ The MS spectrum of 1 also showed a prominent neutral loss of ½M þ 2Na peak at m∕z 773.8754, in agreement with the mole- 237 Da, corresponding to the N-Ac-N-MeTyr appendage on the cular formula C73H107N13O21 (calculated for C73H107N13O21Na, Aound residue (probably lost via a McLafferty-type rearrange- 1524.7597). Thus, metabolite 2 was found to be a homolog of 1, ment; Fig. S6D). By contrast, tychonamide A looses a 187 Da exhibiting homophenylalanine instead of O-MeHty. fragment, in agreement with the cleavage of N-acetyl-N-methyl- leucine (Fig. S6D). Thus, by this independent approach, the iden- Isolation and Structure Determination of Compounds 3 and 4. Liquid tical planar structure elucidation of 1 was assembled.* chromatography using solid-phase extraction cartridges (C18) Next, we turned our attention to the absolute stereochemistry allowed the recovery of 1-4 from the spent medium of a 21- of portoamide A (1), which contained 16 stereocenters. Chiral day culture OSC. However, conversely to what was observed GC-MS analysis of a derivatized sample of 1 revealed the pre- for the biomass, the major compounds were found by LCMS ana- sence of Lð2S;3RÞ-Thr, Dð2R;3SÞ-allo-Ile, LðSÞ-Ser, DðRÞ-Gln lysis to be 3 and 4 (Fig. S2A). The same was observed for the and DðRÞ-O-MeHty, whereas chiral HPLC confirmed the occur- spent medium of a 15-day culture of OSC (Fig. S2D), from which ∼0.03 μg·mL−1 of 1 and 2 were recovered. From HRESIMS, ½M þ Naþ ions at m∕z 1335.6838 for 3 and m∕z 1305.6708 for *During the preparation of this article, binary mixtures determined to contain 1 and 2,as 4 were in agreement with molecular formulae C62H96N12O19 (cal- well as 3 and 4, were published under the name of lyngbyazothrins C, D, and A, B, respectively (42). As mixtures, their NMR data is not directly comparable with the culated for C62H96N12O19Na, 1335.6807) and C61H94N12O18 (cal- NMR data we collected for pure compounds 1 and 2. culated for C61H94N12O18Na, 1305.6701), respectively. In turn,

Leão et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on October 2, 2021 these were consistent with 3 and 4 being the nonesterified ana- Table 1. Inhibitory activity of the mixture of 1 and 2 toward aquatic logues of 1 and 2, respectively, lacking the N-Ac-N-MeTyr moiety photoautrotrophic microorganisms in each case. Likewise, the related compound pahayokolide B was IC50 found to lack the ester-linked N-acetyl-N-methyl-leucine present −1 Phylum Strain (μg·mL ) in pahayokolide A (38). Proof of the relationship of compounds 1 and 3 was obtained from the enzymatic hydrolysis (porcine liver Bacillariophyta esterase) of the ester bond in 1. The resulting hydrolysate con- Cyclotella meneghiniana CCAP 1070∕5 n/a tained a chromatographic peak with a ½M þ Naþ ion at m∕z Chlorophyta 3 Ankistrodesmus falcatus 24.7 1335, which was compared by MS/MS analysis with as extracted Chlamydomonas reinhardtii CCAP 11∕45 12.6 from the spent culture medium. The resulting fragmentation Cyanobacteria patterns deriving from m∕z 1335 ions were identical for both Anabaena sp. LEGE 00233 n/a samples, thus confirming our structural assignment (Fig. S7). Aphanizomenon sp. LEGE 03278 n/a Cylindrospermopsis raciborskii LEGE 28.4 Synergistic Allelopathic Activity of 1 and 2. Pure 1 and 2, a mixture of 99045 1 and 2, and the mixture of compounds extracted from spent Microcystis aeruginosa LEGE 91094 n/a medium were tested for growth inhibitory activity to C. vulgaris n/a—no inhibition observed. in a diverse range of concentrations. Strikingly, neither the pure compounds nor the mixture extracted from the spent medium both in C. vulgaris and the H460 cell line (Fig. 5B). The microalga exhibited appreciable activity. However, the mixture of 1 and 2 was inhibited strongly by a 2∶1 and a 1∶2.6 mixture at exhibited strong activity, completely inhibiting growth of the C. 30 μg·mL−1. The 2∶1 mixture also exhibited a strong cytotoxic −1 −1 −1 vulgaris at 30 μg·mL and having an IC50 of 12.8 μg·mL effect toward H460 cells at 3 μg·mL , while, at this concentra- (Fig. 5A). Similarly, the mixture of compounds 1 and 2 inhibited tion, 4.4∶1 and 1∶2.1 mixtures did not show appreciable cytotoxic the growth of the green microalgae Ankistrodesmus falcatus and activity. Chlamydomonas reinhardtii, as well as the cyanobacterium Cylin- drospermopsis raciborskii (Table 1, Fig. S8). This growth inhibitory Discussion activity was selective to particular microalgae and cyanobacteria; The four metabolites reported herein expand a growing class of for example, the diatom Cyclotella menenghiniana and the cyano- cyanobacterial natural products that include schyzotrin A (40), bacteria Anabaena sp., Aphanizomenon sp., and Microcystis pahayokolides A and B (38), and the tychonamides A and B aeruginosa, were not inhibited by this compound mixture in doses (39). The larger members of these families of metabolites are do- up to 30 μg:mL−1 (Table 1). Finally, we evaluated compounds 1 decapeptides composed of an 11-residue cyclic structure in which and 2 and a mixture of both for cytotoxic activity in the lung the N terminus possesses a doubly modified amino acid linked to cancer H460 cell line and observed a >10-fold increase in the the side chain of a β-amino acid through an ester bond. These potency (IC50) of the mixture when compared to pure 1 or 2 compounds share several conserved residues in their peptidic se- −1 (IC50 for mixture ¼ 0.17 μg·mL compared with 4.08 and quences (Fig. 6). Compounds 1 and 2 combine the α-amino acid −1 1.92 μg·mL , respectively for 1 and 2, Fig. 5A). sequence present in the cyclic portion of the tychonamides with the rare β-amino acid Aound that is present in schyzotrin A and Effect of the Ratio Portoamide A:Portoamide B on the Allelopathic Ac- the pahayokolides. The evolutionary relationships among these tivity. Mixtures with different proportions of 1 and 2 were tested metabolites are thus of considerable interest. With regard to the biosynthetic origin of the aliphatic Aound moiety, its structure implies a new starting unit, possibly 2-hydroxy-4-methylpen- tanoic acid (leucic acid), followed by three malonyl CoA units to generate the required C11 chain. Alternatively, it is possible that this unit derives from a pentaketide in which the initial acetate unit is doubly methylated at its C2 carbon. Precedence for this latter idea is provided by proposals for the polyketide section of the apratoxin series of cyanobacterial metabolite that variably contains an isopropyl or t-butyl terminus in its polyketide section (43). Additional S-adenosylmethionine (SAM) methylation events are likely also responsible for the O- and N- methyl groups in homotyrosine and tyrosine residues, respectively, whereas the acetyl group in this latter residue can be attributed to acetyl-CoA. Because the previously described compounds that are related to the portoamides were isolated as a result of their bactericidal or antitumor activities, their natural role as allelochemicals is strongly plausible. In fact, pahayokolide A was found to moder- ately inhibit a strain of the cyanobacterium Nostoc sp. and several green algae, including Chlorella sp. (44), isolated from the same waterbodyasthepahayokolide-producingLyngbyasp.strain.How- ever,theauthorsdidnotfindactivityamongthecompoundsexuded by this cyanobacterium and were cautious regarding the potential role of this compound as an allelochemical. Interestingly, the Fig. 5. Synergistic activity of 1 and 2.(A) Dose-response curves for C. vulgaris known cyanobacterial allelochemicals discussed in the introduc- growth bioassay (left) and H460 lung cancer cell line cytotoxicity assay (right). — — — 1∶2 6 2∶1 tion are not structurally similar to the compounds reported here. Circles 1; triangles 2; closed squares mixture of 1 and 2 ( . in A, in In our previous screening study for allelochemicals, we used B); open squares—extract from spent medium (composed mainly of 3 and 4). (B) Effect of mixtures with different portoamide A:portoamide B ratios OSC at low cell densities that mimics their likely concentration on their growth inhibitory activity toward C. vulgaris (left) and cytotoxicity in natural water bodies, and under these conditions we observed a toward H460 lung cancer cells (right). Mixtures were tested at 3 μg·mL−1 moderate inhibitory activity toward C. vulgaris (36). Here, we (gray bars) and 30 μg·mL−1 (black bars). partially assign this inhibitory effect to compounds 1 and 2,

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0914343107 Leão et al. Downloaded by guest on October 2, 2021 Fig. 6. Amino acid alignment of cyanobacterial peptides related to 1 (boxes indicate conserved residues among the four metabolites).

and we observed that C. reinhardtii, A. falcatus, and C. raciborskii tems of several gram-positive bacteria, most notably the anthrax may also be susceptible to such allelochemical interactions. The toxin (49). In such systems, proteins are structurally different and ubiquity of all of these various strains of cyanobacteria and mi- play distinct roles and in combination result in the toxic effect. croalgae as well as that of the Oscillatoria genus makes it likely Synergistic mixtures of structurally distinct, co-occurring second- that they encounter one another in natural water systems, and this ary metabolites by some Actinomycetes have been studied in is especially true given the recent geographical expansion of considerable detail (ref. 50 and references therein). Challis C. raciborskii (28). The lower susceptibility of A. falcatus to and Hopwood (50) consider that synergism may be a common the portoamides as compared to C. vulgaris may explain why in- feature of such coproduced secondary metabolites and an impor- hibition of the former was not observed when exposed to OSC tant driving force in their evolution. In the case of 1 and 2, their exudates (36). From the activity of the culture filtrates (Fig. 1A) simultaneous production may have been selected as a result of and the dose-response curve for the exposure of C. vulgaris to 1 their synergistic activity toward an as yet unidentified cellular and 2 (Fig. 5A), one would expect their concentrations in the −1 target or targets. Interestingly, tychonamides A and B (39) also filtrates to be between 1 to 30 μg·mL . However, preliminary differ only in the O-methyl group on the extended aromatic re- attempts to recover and quantify 1 and 2 from the spent medium sidue and may be indicative of an analogous mode of action and of actively growing cultures of OSC have indicated concentra- biological role. The similar activity profiles observed for 1, 2 and tions considerably lower (Fig. S2D). Additionally, the predomi- their binary mixture toward C. vulgaris and the H460 cells, as well as 3 4 nance of the less active metabolites and after extraction of the susceptibility of the cyanobacterium C. raciborskii to the mix- 1 2 the filtrates suggests that the unique ester moiety on and ture, suggest that the targets for these metabolites may be highly is cleaved during or following exudation. Hence, while in the conserved proteins. Moreover, the ester-linked N-Ac-N-MeTyr natural environment the release of the portoamides may have im- seems to be required for activity because the mixture of 3 and 4 CHEMISTRY portant impacts on phytoplankton dynamics, at this time it is not did not exhibit appreciable activity. Because we recovered 3 and possible to fully assign the allelopathic activity of the filtrates to 4 in higher amounts than 1 and 2 from the culture media of these compounds alone. The activity of the filtrates in the early OSC, clues to the natural activity of these metabolites may reside growth stages, in which the cyanobacterial mat is quickly spread- in the study of their conversion. Metabolites 3 and 4 may also ing, suggests that these and other compounds may be used to represent a natural detoxification mechanism, originating from inhibit the growth of nearby organisms that might compete for the spontaneous hydrolytic degradation of the more active meta- substrate and nutrients. Recent evidence (45) suggests that local bolites 1 and 2 in the aqueous medium. ECOLOGY concentrations (i.e., near the producing cells) of allelochemicals In summary, herein we report the isolation and chemical char- may actually be orders of magnitude higher than those in bulk acterization of unique and highly complex secondary metabolites cell-free filtrates. If so, the growth inhibitory effect near OSC that are present in cyanobacterial cells and are released into the cells could be much stronger than predicted from concentrations medium in early growth stages. In natural conditions, the release of compounds 1 and 2 in the filtrates. We would expect the natural ratio of the allelopathic binary mixture to favor portoamide of these substances may impact neighboring phytoplanktonic A, given the larger amount of this metabolite detected during our organisms. The study of the chemical biology of the portoamides chemical exploration of both the cells and culture medium. should lead to a better understanding of the regulation of defense Metabolites from NRPS-derived pathways with modified ami- metabolite production and release in cyanobacteria as well as no acids have the capacity to create an outstanding diversity of their impacts on surrounding biota. natural product structures (46). Cyanobacteria often produce Materials and methods families of such metabolites, differing only in the degree or nature Details regarding strains, culture conditions, bioassays (phytoplanktonic of amino acid modifications (1). The synergism observed for organisms, H460 lung cancer cell line), and bioassay-guided fractionation different mixtures of compounds 1 and 2, in comparison with of OSC are provided in SI Text. The procedures used in the structural elucida- the separated constituents, was unexpected because they differ tion of 1, namely MS/MS comparative dereplication, chiral GC-MS, and chiral only in a single O-methyl group. Synergism in naturally occurring HPLC, as well as compounds 3 and 4, are also detailed in SI Text. mixtures has been observed for the bacteriocins, peptides of ribosomal origin produced by lactic acid bacteria, which in many ACKNOWLEDGMENTS. We thank the assistance of R. Pereira, H. Abreu, and cases have activity only as binary systems (47). These, however, M. Ilarri with the nutrient autoanalyzer, T. Byrum with the lung cancer cell display distinct peptidic sequences. A similar situation was de- line bioassays, O. Vinning with the extraction and VLC procedures, and A. Jansma with the 600 MHz NMR. This work was supported by a PhD scholar- scribed in the synergistic antifungal activity of two classes of cyclic ship to P.N.L. from Fundação para a Ciência e a Tecnologia (SFRH/BD/28771/ peptides obtained from the freshwater cyanobacterium Anabaena 2006) and by National Institutes of Health Grants CA52955, CA100851, and laxa (48). Other examples include the proteic “binary toxin” sys- GM 086283.

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