Synergistic allelochemicals from a freshwater cyanobacterium

P. N. Leao, A. R. Pereira, W.-T. Liu, J. Ng, P. A. Pevzner, P. C. Dorrestein, G. M. Konig, V. M. Vasconcelos, W. H. Gerwick
2010 Proceedings of the National Academy of Sciences of the United States of America  
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
more » ... 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. allelopathy | chemical ecology | Oscillatoria | cyclic peptides | synergism C yanobacteria 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 allelo-
doi:10.1073/pnas.0914343107 pmid:20534563 pmcid:PMC2895120 fatcat:atuzktp3izhfjihg4jiex73ery