Synergistic allelochemicals from a freshwater cyanobacterium

Pedro N. Leão; Alban R. Pereira; Wei-Ting Liu; Julio Ng; Pavel A. Pevzner; Pieter C. Dorrestein; Gabriele M. König; Vitor M. Vasconcelos; William H. Gerwick

doi:10.1073/pnas.0914343107

New Research In

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved May 17, 2010 (received for review December 12, 2009)
↵¹P.N.L. and A.R.P. contributed equally to this work.

Abstract

The ability of cyanobacteria to produce complex secondary metabolites with potent biological activities has gathered considerable attention due to their potential therapeutic and agrochemical applications. However, the precise physiological or ecological roles played by a majority of these metabolites have remained elusive. Several studies have shown that cyanobacteria are able to interfere with other organisms in their communities through the release of compounds into the surrounding medium, a phenomenon usually referred to as allelopathy. Exudates from the freshwater cyanobacterium Oscillatoria sp. had previously been shown to inhibit the green microalga Chlorella vulgaris. In this study, we observed that maximal allelopathic activity is highest in early growth stages of the cyanobacterium, and this provided sufficient material for isolation and chemical characterization of active compounds that inhibited the growth of C. vulgaris. Using a bioassay-guided approach, we isolated and structurally characterized these metabolites as cyclic peptides containing several unusually modified amino acids that are found both in the cells and in the spent media of Oscillatoria sp. cultures. Strikingly, only the mixture of the two most abundant metabolites in the cells was active toward C. vulgaris. Synergism was also observed in a lung cancer cell cytotoxicity assay. The binary mixture inhibited other phytoplanktonic organisms, supporting a natural function of this synergistic mixture of metabolites as allelochemicals.

Cyanobacteria are a prolific source of nearly 800 diverse bioactive secondary metabolites, originating mainly from nonribosomal peptide synthetase (NRPS) or mixed polyketide synthase (PKS)–NRPS biosynthesis (1, 2). Efforts to isolate and characterize cyanobacterial metabolites have usually been motivated from a desire to describe either their natural toxicity toward animals in natural settings (e.g., ref. 3) or promising activities from in vitro biomedical screening programs (e.g., ref. 4, 5). However, in general the ecological role played by the majority of these metabolites is not well known (6, 7). Functions established to date for cyanobacterial secondary metabolites include nitrogen storage (8), UV protection (9), metal chelation (10), defense against predation (11), and quorum sensing (12).

Allelopathy refers to the chemically mediated interaction between plants or microorganisms (13). These interactions are characterized by the release of allelopathic compounds (allelochemicals) into the surrounding medium, eliciting either a positive or deleterious response in a target organism (13). In aquatic ecosystems, allelopathy is regarded as an important process influencing the shaping of microbial communities (14–16). Toxic properties have been attributed to cyanobacteria over the last 130 yr (e.g., ref. 17), and the allelopathic potential of these organisms was described through field-derived observations in the 1970s (18, 19). Since then, several genera of cyanobacteria have been implicated in allelopathic phenomena, with targets ranging from other cyanobacteria to higher plants; unfortunately, only a very limited number of allelochemicals have been identified and mainly from freshwater cyanobacteria (20). These include cyanobacterin, a chlorinated γ-lactone produced by Scytonema hofmanni that inhibits other cyanobacteria and green microalgae (21); fischerellin A, an enediyne-containing photosystem II inhibitor produced by Fischerella muscicula (22); the hapalindoles, small metabolites that have been isolated from Hapalosiphon and Fischerella spp. that inhibit several microorganisms (23, 24); and the nostocyclamides, relatively small cyclic peptides from Nostoc sp. that inhibit cyanobacteria and microalgae (25, 26). Different ecological roles have been attributed to the production of allelochemicals by cyanobacteria, including phytoplankton succession, bloom formation, resource and interference competition (27), and invasive fitness (28). In order to understand the ecological significance of allelopathy in cyanobacteria, several studies have characterized environmental factors that may modulate allelopathic events. The presence of competitors (29–31) and coexisting heterotrophic bacteria that degrade allelochemical substances (32) have been identified among biotic factors. Light intensity (33), temperature (34), nutrient levels, and pH (35) have been shown to control allelochemical production in some species of cyanobacteria.

We had previously reported (36) the inhibitory effect of exudates from the mat-forming cyanobacterium Oscillatoria sp. strain LEGE 05292 (OSC) on the microalga C. vulgaris. Here, we evaluated the influence of abiotic factors and culture conditions on this allelopathic interaction and found that the main factor controlling activity was the growth stage of the OSC cultures. Using bioassay-guided fractionation of the OSC biomass, we isolated one of the compounds responsible for the inhibitory activity (portoamide A, 1) in sufficient quantity for structural characterization by nuclear magnetic resonance (NMR). Confirmation of the structure of 1 and its homolog (portoamide B, 2) was achieved by state-of-the-art mass spectrometric (MS) sequencing methodologies (37). A chemoenzymatic approach together with MS data allowed us to deduce the structures of the related compounds portoamides C and D (3 and 4, respectively). The four metabolites were found to be present both in the cells and in the culture medium of actively growing OSC, thus supporting their role as allelochemicals. 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 growth (IC₅₀ = 12.8 μg·mL^-1). 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 Oscillatoria 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 to inhibit the growth of C. vulgaris. Low-light, high-light, low-temperature, or nutrient limitation did not increase activity when compared to control conditions (Fig. S1). However, when cell densities and culture stage conditions were evaluated, a peak in the inhibitory activity of the filtrates (∼11–27% of growth relative to control) was observed between days 10 and 15 for all cultures, corresponding to the beginning of the exponential growth phase as inferred from nitrate consumption. Filtrates obtained even at very early time points displayed considerable activity (e.g. at 3 d, the filtrate had between 28–89% of growth relative to controls) (Fig. 1).

Fig. 1.

Allelopathic activity of filtrates from OSC grown at different cell densities. (A) Growth of C. vulgaris in the OSC filtrates retrieved at different growth stages. (B) Growth profile of the OSC cultures, as inferred from nitrate removal from the medium.

Bioassay-Guided Isolation and Structural Elucidation of Portoamides A (1) and B (2).

After harvesting and filtration (see SI Text), the lyophilized biomass was repeatedly extracted with CH₂Cl₂/MeOH (2∶1) and fractionated by silica gel chromatography. The most polar fraction (100% methanol) showed high growth inhibitory activity against C. vulgaris and was thus subjected to reverse-phase HPLC to afford cyclic dodecapeptide 1 and its closely related minor analogue 2 (Fig. 2). The smaller metabolites 3 and 4 were also present in this fraction (Fig. S2A) but in insufficient quantity for NMR structural analysis.

Fig. 2.

Major secondary metabolites isolated from OSC biomass or media. Numbering corresponds to portoamide A (1).

The ¹H-NMR spectrum of 1 in DMSO-d₆ (Table S1 and Fig. S3A) displayed a large number of very broad resonances, suggesting both the presence of multiple conformers in solution and a relatively large compound. Differentiated regions for NH protons (δ8.45-7.10), α-methine multiplets (δ5.10-4.10), O-methyl, and N-methyl singlets (δ3.70 and δ2.77 respectively), as well as alkyl chain methyl doublet and triplets (δ0.89-0.80), revealed the peptidic nature of compound 1. Furthermore, numerous resonances between δ7.18-6.50 suggested the occurrence of aromatic amino acid residues. High resolution electrospray ionization mass spectrometry (HRESIMS) analysis yielded an [M + Na]⁺ parent ion at m/z 1554.7695, accompanied by a more intense [M + 2Na]²⁺ peak at m/z 788.8793, confirming a relatively large metabolite structure. Combining this information with NMR data (Table S1), it was possible to derive a molecular formula of C₇₄H₁₀₉N₁₃O₂₂ (calculated for C₇₄H₁₀₉N₁₃O₂₂Na, 1554.7702), exhibiting 27 degrees of unsaturation.

Extensive analysis by 2D NMR, including heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC) (Fig. S4), COSY, and NOESY (Fig. S5), revealed the presence of nine standard and two uncommon amino acids, together with an unusual β-amino acid (Fig. 3A). As shown in Fig. 3A, a combination of COSY and HMBC data led to an unambiguous assembly of spin systems for the following standard amino acid residues: Pro (× 3), Thr, Ser, Ile, Gly, Gln, and Tyr. ¹H and ¹³C chemical shifts for these building blocks were in agreement with literature values (38, 39). Interestingly, Tyr was found to be both methylated and acetylated at its N terminus, as revealed by HMBC correlations from methyl singlets H72 (δ2.77) and H74 (δ1.82) to carbonyl C73 (δ170.4) and α-methine carbon C63 (δ57.6). To our knowledge, this is a previously undescribed combination of appendages detected for Tyr in a cyanobacterial-derived natural product.

Fig. 3.

Selected HMBC, COSY, and NOE correlations for 1 indicating: (A) All the amino acid residues identified. (B) Interresidue connectivity. (N or O: suspected heteroatom attached to the carbonyl group included in the following fragment).

The uncommon amino acid, O-methylhomotyrosine (MeHty), was assembled in analogy to the Tyr residue above. However, an additional methylene (H₂8, δ1.88) was detected as part of the spin system comprised by α-methine H7 (δ4.29) and the benzylic diastereomeric methylene H9 (δ_a2.47 and δ_b2.58). The unique O-methyl singlet (H71, δ3.70) in 1 displayed an HMBC correlation with quaternary carbon C13 (δ157.4) in the 1,4-disubstituted aromatic ring of O-MeHty. Dehydrobutyrine (Dhb) in turn was elucidated from HMBC correlations between the highly diagnostic 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 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 compound 1. Once a good match was found, the observed fragment ions were compared, and mass shifts of differing ions as well as similar mass differences compared to the parent ion allowed rapid identification of identical versus differing portions of these two molecules. In doing so, the algorithm identified that the 46.022 Da mass difference between tychonamide A and compound 1 was located on the Aound unit (see Fig. S6 A and B). An algorithmic description of this procedure is detailed in Ng et al. (37). Finally, with the location of the modified residue in hand, all of the ions in a MS² spectrum were annotated with our annotation script MS-cyclic peptide annotation (MS-CPA) program (41) (Fig. S6C).

Fig. 4 displays a small fraction of representative fragments derived from MALDI-TOF MS² measurements on portoamide A (1). Taken together, fragments A–M supported the linear amino acid sequence Gly-Pro3-Pro2-Ile-Ser-Dhb-Thr-O-MeHty-Pro1-Aound-Gln, confirming the connectivity derived from NOE data. The MS spectrum of 1 also showed a prominent neutral loss of 237 Da, corresponding to the N-Ac-N-MeTyr appendage on the Aound residue (probably lost via a McLafferty-type rearrangement; Fig. S6D). By contrast, tychonamide A looses a 187 Da fragment, in agreement with the cleavage of N-acetyl-N-methylleucine (Fig. S6D). Thus, by this independent approach, the identical planar structure elucidation of 1 was assembled.*

Fig. 4.

Connectivity of amino acid residues in 1, based on selected MALDI-TOF-MS² fragmentations. The cyclic arrangement of the selected fragments is shown (top), together with their respective identification and m/z values (bottom).

Next, we turned our attention to the absolute stereochemistry of portoamide A (1), which contained 16 stereocenters. Chiral GC-MS analysis of a derivatized sample of 1 revealed the presence of L(2S,3R)-Thr, D(2R,3S)-allo-Ile, L(S)-Ser, D(R)-Gln and D(R)-O-MeHty, whereas chiral HPLC confirmed the occurrence of L(S)-Pro and N-Me-L(S)-Tyr (see SI Text). With this, 11 of the 16 stereogenic centers in the planar structure of 1 were assigned. The geometry of the double bond in Dhb was found to be E on the basis of strong NOE correlations between methyl H19 (δ1.09) and C17-NH (δ_H7.76) with methyl H23 (δ1.86) in the double bond.

Portoamide B (2) showed a ¹H NMR (Fig. S3B) very similar to that obtained for portoamide A (1) with the exception of an absent aromatic methoxy singlet at δ3.70 (O-MeHty residue). The absence of a mass equivalent to a -OCH₂- fragment was confirmed by HRESIMS, which displayed an [M + Na]⁺ parent ion at m/z 1524.7541 and was accompanied by an intense [M + 2Na]²⁺ peak at m/z 773.8754, in agreement with the molecular formula C₇₃H₁₀₇N₁₃O₂₁ (calculated for C₇₃H₁₀₇N₁₃O₂₁Na, 1524.7597). Thus, metabolite 2 was found to be a homolog of 1, exhibiting homophenylalanine instead of O-MeHty.

Isolation and Structure Determination of Compounds 3 and 4.

Liquid chromatography using solid-phase extraction cartridges (C₁₈) allowed the recovery of 1-4 from the spent medium of a 21-day culture OSC. However, conversely to what was observed for the biomass, the major compounds were found by LCMS analysis to be 3 and 4 (Fig. S2A). The same was observed for the 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, [M + Na]⁺ ions at m/z 1335.6838 for 3 and m/z 1305.6708 for 4 were in agreement with molecular formulae C₆₂H₉₆N₁₂O₁₉ (calculated for C₆₂H₉₆N₁₂O₁₉Na, 1335.6807) and C₆₁H₉₄N₁₂O₁₈ (calculated for C₆₁H₉₄N₁₂O₁₈Na, 1305.6701), respectively. In turn, these were consistent with 3 and 4 being the nonesterified analogues of 1 and 2, respectively, lacking the N-Ac-N-MeTyr moiety in each case. Likewise, the related compound pahayokolide B was found to lack the ester-linked N-acetyl-N-methyl-leucine present in pahayokolide A (38). Proof of the relationship of compounds 1 and 3 was obtained from the enzymatic hydrolysis (porcine liver esterase) of the ester bond in 1. The resulting hydrolysate contained a chromatographic peak with a [M + Na]⁺ ion at m/z 1335, which was compared by MS/MS analysis with 3 as extracted from the spent culture medium. The resulting fragmentation patterns deriving from m/z 1335 ions were identical for both samples, thus confirming our structural assignment (Fig. S7).

Synergistic Allelopathic Activity of 1 and 2.

Pure 1 and 2, a mixture of 1 and 2, and the mixture of compounds extracted from spent medium were tested for growth inhibitory activity to C. vulgaris in a diverse range of concentrations. Strikingly, neither the pure compounds nor the mixture extracted from the spent medium exhibited appreciable activity. However, the mixture of 1 and 2 exhibited strong activity, completely inhibiting growth of the C. vulgaris at 30 μg·mL^-1 and having an IC₅₀ of 12.8 μg·mL^-1 (Fig. 5A). Similarly, the mixture of compounds 1 and 2 inhibited the growth of the green microalgae Ankistrodesmus falcatus and Chlamydomonas reinhardtii, as well as the cyanobacterium Cylindrospermopsis raciborskii (Table 1, Fig. S8). This growth inhibitory activity was selective to particular microalgae and cyanobacteria; for example, the diatom Cyclotella menenghiniana and the cyanobacteria Anabaena sp., Aphanizomenon sp., and Microcystis aeruginosa, were not inhibited by this compound mixture in doses up to 30 μg.mL^-1 (Table 1). Finally, we evaluated compounds 1 and 2 and a mixture of both for cytotoxic activity in the lung cancer H460 cell line and observed a > 10-fold increase in the potency (IC₅₀) of the mixture when compared to pure 1 or 2 (IC₅₀ for mixture = 0.17 μg·mL^-1 compared with 4.08 and 1.92 μg·mL^-1, respectively for 1 and 2, Fig. 5A).

Fig. 5.

Synergistic activity of 1 and 2. (A) Dose-response curves for C. vulgaris growth bioassay (left) and H460 lung cancer cell line cytotoxicity assay (right). Circles—1; triangles—2; closed squares—mixture of 1 and 2 (1∶2.6 in A, 2∶1 in B); open squares—extract from spent medium (composed mainly of 3 and 4). (B) Effect of mixtures with different portoamide A:portoamide B ratios on their growth inhibitory activity toward C. vulgaris (left) and cytotoxicity toward H460 lung cancer cells (right). Mixtures were tested at 3 μg·mL^-1 (gray bars) and 30 μg·mL^-1 (black bars).

View this table:

Table 1.

Inhibitory activity of the mixture of 1 and 2 toward aquatic photoautrotrophic microorganisms

Effect of the Ratio Portoamide A:Portoamide B on the Allelopathic Activity.

Mixtures with different proportions of 1 and 2 were tested both in C. vulgaris and the H460 cell line (Fig. 5B). The microalga was inhibited strongly by a 2∶1 and a 1∶2.6 mixture at 30 μg·mL^-1. The 2∶1 mixture also exhibited a strong cytotoxic effect toward H460 cells at 3 μg·mL^-1, while, at this concentration, 4.4∶1 and 1∶2.1 mixtures did not show appreciable cytotoxic activity.

Discussion

The four metabolites reported herein expand a growing class of cyanobacterial natural products that include schyzotrin A (40), pahayokolides A and B (38), and the tychonamides A and B (39). The larger members of these families of metabolites are dodecapeptides composed of an 11-residue cyclic structure in which the N terminus possesses a doubly modified amino acid linked to the side chain of a β-amino acid through an ester bond. These compounds share several conserved residues in their peptidic sequences (Fig. 6). Compounds 1 and 2 combine the α-amino acid sequence present in the cyclic portion of the tychonamides with the rare β-amino acid Aound that is present in schyzotrin A and the pahayokolides. The evolutionary relationships among these 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-methylpentanoic 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.

Fig. 6.

Amino acid alignment of cyanobacterial peptides related to 1 (boxes indicate conserved residues among the four metabolites).

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 moderately inhibit a strain of the cyanobacterium Nostoc sp. and several green algae, including Chlorella sp. (44), isolated from the same water body as the pahayokolide-producing Lyngbya sp. strain. However, the authors did not find activity among the compounds exuded by this cyanobacterium and were cautious regarding the potential role of this compound as an allelochemical. Interestingly, the known cyanobacterial allelochemicals discussed in the introduction are not structurally similar to the compounds reported here.

In our previous screening study for allelochemicals, we used OSC at low cell densities that mimics their likely concentration in natural water bodies, and under these conditions we observed a moderate inhibitory activity toward C. vulgaris (36). Here, we partially assign this inhibitory effect to compounds 1 and 2, and we observed that C. reinhardtii, A. falcatus, and C. raciborskii may also be susceptible to such allelochemical interactions. The ubiquity of all of these various strains of cyanobacteria and microalgae as well as that of the Oscillatoria genus makes it likely that they encounter one another in natural water systems, and this is especially true given the recent geographical expansion of C. raciborskii (28). The lower susceptibility of A. falcatus to the portoamides as compared to C. vulgaris may explain why inhibition of the former was not observed when exposed to OSC exudates (36). From the activity of the culture filtrates (Fig. 1A) and the dose-response curve for the exposure of C. vulgaris to 1 and 2 (Fig. 5A), one would expect their concentrations in the filtrates to be between 1 to 30 μg·mL^-1. However, preliminary attempts to recover and quantify 1 and 2 from the spent medium of actively growing cultures of OSC have indicated concentrations considerably lower (Fig. S2D). Additionally, the predominance of the less active metabolites 3 and 4 after extraction of the filtrates suggests that the unique ester moiety on 1 and 2 is cleaved during or following exudation. Hence, while in the natural environment the release of the portoamides may have important impacts on phytoplankton dynamics, at this time it is not possible to fully assign the allelopathic activity of the filtrates to these compounds alone. The activity of the filtrates in the early growth stages, in which the cyanobacterial mat is quickly spreading, suggests that these and other compounds may be used to inhibit the growth of nearby organisms that might compete for substrate and nutrients. Recent evidence (45) suggests that local concentrations (i.e., near the producing cells) of allelochemicals may actually be orders of magnitude higher than those in bulk cell-free filtrates. If so, the growth inhibitory effect near OSC cells could be much stronger than predicted from concentrations of compounds 1 and 2 in the filtrates. We would expect the natural ratio of the allelopathic binary mixture to favor portoamide A, given the larger amount of this metabolite detected during our chemical exploration of both the cells and culture medium.

Metabolites from NRPS-derived pathways with modified amino acids have the capacity to create an outstanding diversity of natural product structures (46). Cyanobacteria often produce families of such metabolites, differing only in the degree or nature of amino acid modifications (1). The synergism observed for different mixtures of compounds 1 and 2, in comparison with the separated constituents, was unexpected because they differ only in a single O-methyl group. Synergism in naturally occurring mixtures has been observed for the bacteriocins, peptides of ribosomal origin produced by lactic acid bacteria, which in many cases have activity only as binary systems (47). These, however, display distinct peptidic sequences. A similar situation was described in the synergistic antifungal activity of two classes of cyclic peptides obtained from the freshwater cyanobacterium Anabaena laxa (48). Other examples include the proteic “binary toxin” systems of several gram-positive bacteria, most notably the anthrax toxin (49). In such systems, proteins are structurally different and play distinct roles and in combination result in the toxic effect. Synergistic mixtures of structurally distinct, co-occurring secondary metabolites by some Actinomycetes have been studied in considerable detail (ref. 50 and references therein). Challis and Hopwood (50) consider that synergism may be a common feature of such coproduced secondary metabolites and an important driving force in their evolution. In the case of 1 and 2, their simultaneous production may have been selected as a result of their synergistic activity toward an as yet unidentified cellular target or targets. Interestingly, tychonamides A and B (39) also differ only in the O-methyl group on the extended aromatic residue and may be indicative of an analogous mode of action and biological role. The similar activity profiles observed for 1, 2 and their binary mixture toward C. vulgaris and the H460 cells, as well as the susceptibility of the cyanobacterium C. raciborskii to the mixture, suggest that the targets for these metabolites may be highly conserved proteins. Moreover, the ester-linked N-Ac-N-MeTyr seems to be required for activity because the mixture of 3 and 4 did not exhibit appreciable activity. Because we recovered 3 and 4 in higher amounts than 1 and 2 from the culture media of OSC, clues to the natural activity of these metabolites may reside in the study of their conversion. Metabolites 3 and 4 may also represent a natural detoxification mechanism, originating from the spontaneous hydrolytic degradation of the more active metabolites 1 and 2 in the aqueous medium.

In summary, herein we report the isolation and chemical characterization of unique and highly complex secondary metabolites that are present in cyanobacterial cells and are released into the medium in early growth stages. In natural conditions, the release of these substances may impact neighboring phytoplanktonic organisms. The study of the chemical biology of the portoamides should lead to a better understanding of the regulation of defense metabolite production and release in cyanobacteria as well as their impacts on surrounding biota.

Materials and methods

Details regarding strains, culture conditions, bioassays (phytoplanktonic organisms, H460 lung cancer cell line), and bioassay-guided fractionation of OSC are provided in SI Text. The procedures used in the structural elucidation of 1, namely MS/MS comparative dereplication, chiral GC-MS, and chiral HPLC, as well as compounds 3 and 4, are also detailed in SI Text.

Acknowledgments

We thank the assistance of R. Pereira, H. Abreu, and M. Ilarri with the nutrient autoanalyzer, T. Byrum with the lung cancer cell 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 scholarship to P.N.L. from Fundação para a Ciência e a Tecnologia (SFRH/BD/28771/2006) and by National Institutes of Health Grants CA52955, CA100851, and GM 086283.

Footnotes

²To whom correspondence may be addressed. E-mail: vmvascon{at}fc.up.pt and wgerwick{at}ucsd.edu.
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., 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 reagents/analytic tools; and P.N.L., A.R.P., V.M.V., and W.H.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0914343107/-/DCSupplemental.
Data deposition: The DNA sequence reported in this paper was deposited in the GenBank database (accession no. GU085101)
↵^*During the preparation of this article, binary mixtures determined to contain 1 and 2, as 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 NMR data we collected for pure compounds 1 and 2.

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2. Paul VJ
(1999) Production of secondary metabolites by filamentous tropical marine cyanobacteria: Ecological functions of the compounds. J Phycol 35(6):1412–1421.
OpenUrl CrossRef
↵
1. Sharif DI,
2. Gallon J,
3. Smith CJ,
4. Dudley E
(2008) Quorum sensing in Cyanobacteria: N-octanoyl-homoserine lactone release and response, by the epilithic colonial cyanobacterium Gloeothece PCC6909. Isme J 2(12):1171–1182.
OpenUrl CrossRef PubMed
↵
1. Rice EL
(1984) Allelopathy (Academic, New York), 2nd Ed.
↵
1. Gross EM
(2003) Allelopathy of aquatic autotrophs. Crit Rev Plant Sci 22(3&4):313–339.
OpenUrl CrossRef
↵
1. Strom SL
(2008) Microbial ecology of ocean biogeochemistry: A community perspective. Science 320(5879):1043–1045.
OpenUrl Abstract/FREE Full Text
↵
1. Hay ME
(2009) Marine chemical ecology: Chemical signals and cues structure marine populations, communities, and ecosystems. Annu Rev Mar Sci 1:193–212.
OpenUrl CrossRef
↵
1. Francis G
(1878) Poisonous Australian lake. Nature 18:11–12.
OpenUrl
↵
1. Keating KI
(1977) Allelopathic influence on blue-green bloom sequence in a eutrophic lake. Science 196(4292):885–887.
OpenUrl Abstract/FREE Full Text
↵
1. Keating KI
(1978) Blue-green-algal inhibition of diatom growth—Transition from mesotrophic to eutrophic community structure. Science 199(4332):971–973.
OpenUrl Abstract/FREE Full Text
↵
1. Leão PN,
2. Vasconcelos MTSD,
3. Vasconcelos VM
(2009) Allelopathy in freshwater cyanobacteria. Crit Rev Microbiol 35(4):271–282.
OpenUrl CrossRef PubMed
↵
1. Mason CP,
2. et al.
(1982) Isolation of chlorine-containing antibiotic from the freshwater cyanobacterium Scytonema hofmanni. Science 213(4531):400–402.
OpenUrl
↵
1. Hagmann L,
2. Jüttner F
(1996) Fischerellin A: A novel photosystem-II-inhibiting allelochemical of the cyanobacterium Fischerella muscicola with antifungal and herbicidal activity. Tetrahedron Lett 37(36):6539–6542.
OpenUrl CrossRef
↵
1. Moore RE,
2. Cheuk C,
3. Patterson GML
(1984) Hapalindoles: New alkaloids from the blue-green alga Hapalosiphon fontinalis. J Am Chem Soc 106:6456–6457.
OpenUrl CrossRef
↵
1. Doan NT,
2. Rickards RW,
3. Rothschild JM,
4. Smith GD
(2000) Allelopathic actions of the alkaloid 12-epi-hapalindole E isonitrile and calothrixin A from cyanobacteria of the genera Fischerella and Calothrix. J Appl Phycol 12:409–416.
OpenUrl CrossRef
↵
1. Todorova AK,
2. Jüttner F
(1995) Nostocyclamide: A new macrocyclic, thiazole-containing allelochemical from Nostoc sp. 31 (Cyanobacteria) J Org Chem 60:7891–7895.
OpenUrl CrossRef
↵
1. Jüttner F,
2. Todorova AK,
3. Walch N,
4. Philipsborn Wv
(2001) Nostocyclamide M: A cyanobacterial cyclic peptide with allelopathic activity from Nostoc 31. Phytochemistry 57:613–619.
OpenUrl CrossRef PubMed
↵
1. Leflaive J,
2. Ten-Hage L
(2007) Algal and cyanobacterial secondary metabolites in freshwaters: A comparison of allelopathic compounds and toxins. Freshwater Biol 52(2):199–214.
OpenUrl CrossRef
↵
1. Figueredo CC,
2. Giani A,
3. Bird DF
(2007) Does allelopathy contribute to Cylindrospermopsis raciborskii (cyanobacteria) bloom occurrence and geographic expansion? J Phycol 43(2):256–265.
OpenUrl CrossRef
↵
1. Jang MH,
2. Ha K,
3. Takamura N
(2007) Reciprocal allelopathic responses between toxic cyanobacteria (Microcystis aeruginosa) and duckweed (Lemna japonica) Toxicon 49:727–733.
OpenUrl PubMed
↵
1. Vardi A,
2. et al.
(2002) Dinoflagellate-cyanobacterium communication may determine the composition of phytoplankton assemblage in a mesotrophic lake. Curr Biol 12:1767–1772.
OpenUrl CrossRef PubMed
↵
1. Kearns KD,
2. Hunter MD
(2000) Green algal extracellular products regulate antialgal toxin production in a cyanobacterium. Environ Microbiol 2(3):291–297.
OpenUrl CrossRef PubMed
↵
1. Hulot FD,
2. Huisman J
(2004) Allelopathic interactions between phytoplankton species: The roles of heterotrophic bacteria and mixing intensity. Limnol Oceanogr 49(4):1424–1434.
OpenUrl CrossRef
↵
1. De Nobel WT,
2. Matthijs HCP,
3. Von Elert E,
4. Mur LR
(1998) Comparison of the light-limited growth of the nitrogen-fixing cyanobacteria Anabaena and Aphanizomenon. New Phytol 138(4):579–587.
OpenUrl CrossRef
↵
1. de Figueiredo DR,
2. et al.
(2006) The effect of environmental parameters and cyanobacterial blooms on phytoplankton dynamics of a Portuguese temperate lake. Hydrobiologia 568:145–157.
OpenUrl CrossRef
↵
1. Ray S,
2. Bagchi SN
(2001) Nutrients and pH regulate algicide accumulation in cultures of the cyanobacterium Oscillatoria laetevirens. New Phytol 149:455–460.
OpenUrl CrossRef
↵
1. Leão PN,
2. Vasconcelos MTSD,
3. Vasconcelos VM
(2009) Alleopathic activity of cyanobacteria on green microalgae at low cell densities. Eur J Phycol 44(3):347–355.
OpenUrl CrossRef
↵
1. Ng J,
2. et al.
(2009) Dereplication and de novo sequencing of nonribosomal peptides. Nat Methods 6(8):596–599.
OpenUrl CrossRef PubMed
↵
1. An TY,
2. et al.
(2007) Structures of pahayokolides A and B, cyclic peptides from a Lyngbya sp. J Nat Prod 70(5):730–735.
OpenUrl PubMed
↵
1. Mehner C,
2. et al.
(2008) A novel beta-amino acid in cytotoxic peptides from the cyanobacterium Tychonema sp. Eur J Org Chem 2008(10):1732–1739.
OpenUrl CrossRef
↵
1. Pergament I,
2. Carmeli S
(1994) Schizotrin-A—A novel antimicrobial cyclic peptide from a cyanobacterium. Tetrahedron Lett 35(45):8473–8476.
OpenUrl CrossRef
↵
1. Liu WT,
2. et al.
(2009) Interpretation of tandem mass spectra obtained from cyclic nonribosomal peptides. Anal Chem 81(11):4200–4209.
OpenUrl CrossRef PubMed
↵
1. Zainuddin EN,
2. et al.
(2009) Lyngbyazothrins A-D antimicrobial cyclic undecapeptides from the cultured cyanobacterium Lyngbya sp. J Nat Prod 72(8):1373–1378.
OpenUrl PubMed
↵
1. Luesch H,
2. Yoshida WY,
3. Moore RE,
4. Paul VJ
(2002) New apratoxins of marine cyanobacterial origin from Guam and Palau. Bioorg Med Chem 10(6):1973–1978.
OpenUrl CrossRef PubMed
↵
1. Berry JP,
2. Gantar M,
3. Gawley RE,
4. Wang M,
5. Rein KS
(2004) Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades. Comp Biochem Physiol, Part C: Toxicol Pharmacol 139(4):231–238.
OpenUrl CrossRef
↵
1. Jonsson PR,
2. Pavia H,
3. Toth G
(2009) Formation of harmful algal blooms cannot be explained by allelopathic interactions. Proc Natl Acad Sci USA 106(27):11177–11182.
OpenUrl Abstract/FREE Full Text
↵
1. Fischbach MA,
2. Walsh CT
(2006) Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms. Chem Rev 106(8):3468–3496.
OpenUrl CrossRef PubMed
↵
1. Garneau S,
2. Martin NI,
3. Vederas JC
(2002) Two-peptide bacteriocins produced by lactic acid bacteria. Biochimie 84(5–6):577–592.
OpenUrl CrossRef PubMed
↵
1. Frankmolle WP,
2. et al.
(1992) Antifungal cyclic-peptides from the terrestrial blue-green-alga Anabaena laxa. 1. Isolation and biological properties. Jpn J Antibiot 45(9):1451–1457.
OpenUrl PubMed
↵
1. Barth H,
2. Aktories K,
3. Popoff MR,
4. Stiles BG
(2004) Binary bacterial toxins: Biochemistry, biology, and applications of common Clostridium and Bacillus proteins. Microbiol Mol Biol R 68(3):373–402.
OpenUrl Abstract/FREE Full Text
↵
1. Challis GL,
2. Hopwood DA
(2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci USA 100:14555–14561.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 107 (25)

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Submit

Article Classifications

Physical Sciences
Chemistry

Biological Sciences
Ecology

[1] ↵
Welker M,
von Dohren H
(2006) Cyanobacterial peptides—Nature’s own combinatorial biosynthesis. FEMS Microbiol Rev 30(4):530–563.
OpenUrl CrossRef PubMed

[2] Welker M,

[3] von Dohren H

[4] ↵
Tan LT
(2007) Bioactive natural products from marine cyanobacteria for drug discovery. Phytochemistry 68(7):954–979.
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[5] Tan LT

[6] ↵
Rinehart KL,
et al.
(1988) Nodularin, Microcystin, and the configuration of Adda. J Am Chem Soc 110(25):8557–8558.
OpenUrl CrossRef

[7] Rinehart KL,

[8] et al.

[9] ↵
Edwards DJ,
et al.
(2004) Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula. Chem Biol 11(6):817–833.
OpenUrl CrossRef PubMed

[10] Edwards DJ,

[11] et al.

[12] ↵
Han BN,
Gross H,
Goeger DE,
Mooberry SL,
Gerwick WH
(2006) Aurilides B and C, cancer cell toxins from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula. J Nat Prod 69(4):572–575.
OpenUrl PubMed

[13] Han BN,

[14] Gross H,

[15] Goeger DE,

[16] Mooberry SL,

[17] Gerwick WH

[18] ↵
Babica P,
Blaha L,
Marsalek B
(2006) Exploring the natural role of microcystins—A review of effects on photoautotrophic organisms. J Phycol 42:9–20.
OpenUrl CrossRef

[19] Babica P,

[20] Blaha L,

[21] Marsalek B

[22] ↵
Schatz D,
et al.
(2007) Towards clarification of the biological role of microcystins, a family of cyanobacterial toxins. Environ Microbiol 9(4):965–970.
OpenUrl CrossRef PubMed

[23] Schatz D,

[24] et al.

[25] ↵
Simon RD
(1971) Cyanophycin granules from blue-green alga Anabaena cylindrica—Reserve material consisting of copolymers of aspartic acid and arginine. Proc Natl Acad Sci USA 68(2):265–267.
OpenUrl Abstract/FREE Full Text

[26] Simon RD

[27] ↵
Garciapichel F,
Sherry ND,
Castenholz RW
(1992) Evidence for an ultraviolet sunscreen role of the extracellular pigment scytonemin in the terrestrial cyanobacterium Chlorogloeopsis sp. Photochem Photobiol 56(1):17–23.
OpenUrl CrossRef PubMed

[28] Garciapichel F,

[29] Sherry ND,

[30] Castenholz RW

[31] ↵
Itou Y,
Okada S,
Murakami M
(2001) Two structural isomeric siderophores from the freshwater cyanobacterium Anabaena cylindrica (NIES-19) Tetrahedron 57(44):9093–9099.
OpenUrl CrossRef

[32] Itou Y,

[33] Okada S,

[34] Murakami M

[35] ↵
Nagle DG,
Paul VJ
(1999) Production of secondary metabolites by filamentous tropical marine cyanobacteria: Ecological functions of the compounds. J Phycol 35(6):1412–1421.
OpenUrl CrossRef

[36] Nagle DG,

[37] Paul VJ

[38] ↵
Sharif DI,
Gallon J,
Smith CJ,
Dudley E
(2008) Quorum sensing in Cyanobacteria: N-octanoyl-homoserine lactone release and response, by the epilithic colonial cyanobacterium Gloeothece PCC6909. Isme J 2(12):1171–1182.
OpenUrl CrossRef PubMed

[39] Sharif DI,

[40] Gallon J,

[41] Smith CJ,

[42] Dudley E

[43] ↵
Rice EL
(1984) Allelopathy (Academic, New York), 2nd Ed.

[44] Rice EL

[45] ↵
Gross EM
(2003) Allelopathy of aquatic autotrophs. Crit Rev Plant Sci 22(3&4):313–339.
OpenUrl CrossRef

[46] Gross EM

[47] ↵
Strom SL
(2008) Microbial ecology of ocean biogeochemistry: A community perspective. Science 320(5879):1043–1045.
OpenUrl Abstract/FREE Full Text

[48] Strom SL

[49] ↵
Hay ME
(2009) Marine chemical ecology: Chemical signals and cues structure marine populations, communities, and ecosystems. Annu Rev Mar Sci 1:193–212.
OpenUrl CrossRef

[50] Hay ME

[51] ↵
Francis G
(1878) Poisonous Australian lake. Nature 18:11–12.
OpenUrl

[52] Francis G

[53] ↵
Keating KI
(1977) Allelopathic influence on blue-green bloom sequence in a eutrophic lake. Science 196(4292):885–887.
OpenUrl Abstract/FREE Full Text

[54] Keating KI

[55] ↵
Keating KI
(1978) Blue-green-algal inhibition of diatom growth—Transition from mesotrophic to eutrophic community structure. Science 199(4332):971–973.
OpenUrl Abstract/FREE Full Text

[56] Keating KI

[57] ↵
Leão PN,
Vasconcelos MTSD,
Vasconcelos VM
(2009) Allelopathy in freshwater cyanobacteria. Crit Rev Microbiol 35(4):271–282.
OpenUrl CrossRef PubMed

[58] Leão PN,

[59] Vasconcelos MTSD,

[60] Vasconcelos VM

[61] ↵
Mason CP,
et al.
(1982) Isolation of chlorine-containing antibiotic from the freshwater cyanobacterium Scytonema hofmanni. Science 213(4531):400–402.
OpenUrl

[62] Mason CP,

[63] et al.

[64] ↵
Hagmann L,
Jüttner F
(1996) Fischerellin A: A novel photosystem-II-inhibiting allelochemical of the cyanobacterium Fischerella muscicola with antifungal and herbicidal activity. Tetrahedron Lett 37(36):6539–6542.
OpenUrl CrossRef

[65] Hagmann L,

[66] Jüttner F

[67] ↵
Moore RE,
Cheuk C,
Patterson GML
(1984) Hapalindoles: New alkaloids from the blue-green alga Hapalosiphon fontinalis. J Am Chem Soc 106:6456–6457.
OpenUrl CrossRef

[68] Moore RE,

[69] Cheuk C,

[70] Patterson GML

[71] ↵
Doan NT,
Rickards RW,
Rothschild JM,
Smith GD
(2000) Allelopathic actions of the alkaloid 12-epi-hapalindole E isonitrile and calothrixin A from cyanobacteria of the genera Fischerella and Calothrix. J Appl Phycol 12:409–416.
OpenUrl CrossRef

[72] Doan NT,

[73] Rickards RW,

[74] Rothschild JM,

[75] Smith GD

[76] ↵
Todorova AK,
Jüttner F
(1995) Nostocyclamide: A new macrocyclic, thiazole-containing allelochemical from Nostoc sp. 31 (Cyanobacteria) J Org Chem 60:7891–7895.
OpenUrl CrossRef

[77] Todorova AK,

[78] Jüttner F

[79] ↵
Jüttner F,
Todorova AK,
Walch N,
Philipsborn Wv
(2001) Nostocyclamide M: A cyanobacterial cyclic peptide with allelopathic activity from Nostoc 31. Phytochemistry 57:613–619.
OpenUrl CrossRef PubMed

[80] Jüttner F,

[81] Todorova AK,

[82] Walch N,

[83] Philipsborn Wv

[84] ↵
Leflaive J,
Ten-Hage L
(2007) Algal and cyanobacterial secondary metabolites in freshwaters: A comparison of allelopathic compounds and toxins. Freshwater Biol 52(2):199–214.
OpenUrl CrossRef

[85] Leflaive J,

[86] Ten-Hage L

[87] ↵
Figueredo CC,
Giani A,
Bird DF
(2007) Does allelopathy contribute to Cylindrospermopsis raciborskii (cyanobacteria) bloom occurrence and geographic expansion? J Phycol 43(2):256–265.
OpenUrl CrossRef

[88] Figueredo CC,

[89] Giani A,

[90] Bird DF

[91] ↵
Jang MH,
Ha K,
Takamura N
(2007) Reciprocal allelopathic responses between toxic cyanobacteria (Microcystis aeruginosa) and duckweed (Lemna japonica) Toxicon 49:727–733.
OpenUrl PubMed

[92] Jang MH,

[93] Ha K,

[94] Takamura N

[95] ↵
Vardi A,
et al.
(2002) Dinoflagellate-cyanobacterium communication may determine the composition of phytoplankton assemblage in a mesotrophic lake. Curr Biol 12:1767–1772.
OpenUrl CrossRef PubMed

[96] Vardi A,

[97] et al.

[98] ↵
Kearns KD,
Hunter MD
(2000) Green algal extracellular products regulate antialgal toxin production in a cyanobacterium. Environ Microbiol 2(3):291–297.
OpenUrl CrossRef PubMed

[99] Kearns KD,

[100] Hunter MD

[101] ↵
Hulot FD,
Huisman J
(2004) Allelopathic interactions between phytoplankton species: The roles of heterotrophic bacteria and mixing intensity. Limnol Oceanogr 49(4):1424–1434.
OpenUrl CrossRef

[102] Hulot FD,

[103] Huisman J

[104] ↵
De Nobel WT,
Matthijs HCP,
Von Elert E,
Mur LR
(1998) Comparison of the light-limited growth of the nitrogen-fixing cyanobacteria Anabaena and Aphanizomenon. New Phytol 138(4):579–587.
OpenUrl CrossRef

[105] De Nobel WT,

[106] Matthijs HCP,

[107] Von Elert E,

[108] Mur LR

[109] ↵
de Figueiredo DR,
et al.
(2006) The effect of environmental parameters and cyanobacterial blooms on phytoplankton dynamics of a Portuguese temperate lake. Hydrobiologia 568:145–157.
OpenUrl CrossRef

[110] de Figueiredo DR,

[111] et al.

[112] ↵
Ray S,
Bagchi SN
(2001) Nutrients and pH regulate algicide accumulation in cultures of the cyanobacterium Oscillatoria laetevirens. New Phytol 149:455–460.
OpenUrl CrossRef

[113] Ray S,

[114] Bagchi SN

[115] ↵
Leão PN,
Vasconcelos MTSD,
Vasconcelos VM
(2009) Alleopathic activity of cyanobacteria on green microalgae at low cell densities. Eur J Phycol 44(3):347–355.
OpenUrl CrossRef

[116] Leão PN,

[117] Vasconcelos MTSD,

[118] Vasconcelos VM

[119] ↵
Ng J,
et al.
(2009) Dereplication and de novo sequencing of nonribosomal peptides. Nat Methods 6(8):596–599.
OpenUrl CrossRef PubMed

[120] Ng J,

[121] et al.

[122] ↵
An TY,
et al.
(2007) Structures of pahayokolides A and B, cyclic peptides from a Lyngbya sp. J Nat Prod 70(5):730–735.
OpenUrl PubMed

[123] An TY,

[124] et al.

[125] ↵
Mehner C,
et al.
(2008) A novel beta-amino acid in cytotoxic peptides from the cyanobacterium Tychonema sp. Eur J Org Chem 2008(10):1732–1739.
OpenUrl CrossRef

[126] Mehner C,

[127] et al.

[128] ↵
Pergament I,
Carmeli S
(1994) Schizotrin-A—A novel antimicrobial cyclic peptide from a cyanobacterium. Tetrahedron Lett 35(45):8473–8476.
OpenUrl CrossRef

[129] Pergament I,

[130] Carmeli S

[131] ↵
Liu WT,
et al.
(2009) Interpretation of tandem mass spectra obtained from cyclic nonribosomal peptides. Anal Chem 81(11):4200–4209.
OpenUrl CrossRef PubMed

[132] Liu WT,

[133] et al.

[134] ↵
Zainuddin EN,
et al.
(2009) Lyngbyazothrins A-D antimicrobial cyclic undecapeptides from the cultured cyanobacterium Lyngbya sp. J Nat Prod 72(8):1373–1378.
OpenUrl PubMed

[135] Zainuddin EN,

[136] et al.

[137] ↵
Luesch H,
Yoshida WY,
Moore RE,
Paul VJ
(2002) New apratoxins of marine cyanobacterial origin from Guam and Palau. Bioorg Med Chem 10(6):1973–1978.
OpenUrl CrossRef PubMed

[138] Luesch H,

[139] Yoshida WY,

[140] Moore RE,

[141] Paul VJ

[142] ↵
Berry JP,
Gantar M,
Gawley RE,
Wang M,
Rein KS
(2004) Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades. Comp Biochem Physiol, Part C: Toxicol Pharmacol 139(4):231–238.
OpenUrl CrossRef

[143] Berry JP,

[144] Gantar M,

[145] Gawley RE,

[146] Wang M,

[147] Rein KS

[148] ↵
Jonsson PR,
Pavia H,
Toth G
(2009) Formation of harmful algal blooms cannot be explained by allelopathic interactions. Proc Natl Acad Sci USA 106(27):11177–11182.
OpenUrl Abstract/FREE Full Text

[149] Jonsson PR,

[150] Pavia H,

[151] Toth G

[152] ↵
Fischbach MA,
Walsh CT
(2006) Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms. Chem Rev 106(8):3468–3496.
OpenUrl CrossRef PubMed

[153] Fischbach MA,

[154] Walsh CT

[155] ↵
Garneau S,
Martin NI,
Vederas JC
(2002) Two-peptide bacteriocins produced by lactic acid bacteria. Biochimie 84(5–6):577–592.
OpenUrl CrossRef PubMed

[156] Garneau S,

[157] Martin NI,

[158] Vederas JC

[159] ↵
Frankmolle WP,
et al.
(1992) Antifungal cyclic-peptides from the terrestrial blue-green-alga Anabaena laxa. 1. Isolation and biological properties. Jpn J Antibiot 45(9):1451–1457.
OpenUrl PubMed

[160] Frankmolle WP,

[161] et al.

[162] ↵
Barth H,
Aktories K,
Popoff MR,
Stiles BG
(2004) Binary bacterial toxins: Biochemistry, biology, and applications of common Clostridium and Bacillus proteins. Microbiol Mol Biol R 68(3):373–402.
OpenUrl Abstract/FREE Full Text

[163] Barth H,

[164] Aktories K,

[165] Popoff MR,

[166] Stiles BG

[167] ↵
Challis GL,
Hopwood DA
(2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci USA 100:14555–14561.
OpenUrl Abstract/FREE Full Text

[168] Challis GL,

[169] Hopwood DA

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New Research In

Synergistic allelochemicals from a freshwater cyanobacterium

Abstract

Results

Identification of the Producing Strain (OSC).

Influence of Biotic and Abiotic Factors on Allelopathic Activity.

Bioassay-Guided Isolation and Structural Elucidation of Portoamides A (1) and B (2).

Isolation and Structure Determination of Compounds 3 and 4.

Synergistic Allelopathic Activity of 1 and 2.

Effect of the Ratio Portoamide A:Portoamide B on the Allelopathic Activity.

Discussion

Materials and methods

Acknowledgments

Footnotes

References

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Synergistic allelochemicals from a freshwater cyanobacterium

Abstract

Results

Identification of the Producing Strain (OSC).

Influence of Biotic and Abiotic Factors on Allelopathic Activity.

Bioassay-Guided Isolation and Structural Elucidation of Portoamides A (1) and B (2).

Isolation and Structure Determination of Compounds 3 and 4.

Synergistic Allelopathic Activity of 1 and 2.

Effect of the Ratio Portoamide A:Portoamide B on the Allelopathic Activity.

Discussion

Materials and methods

Acknowledgments

Footnotes

References

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