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Global Metabolomic Characterizations of Microcystis Spp. Highlights Clonal Diversity in Natural Bloom-Forming Populations and Expands Metabolite Structural Diversity

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Global Metabolomic Characterizations of Microcystis Spp. Highlights Clonal Diversity in Natural Bloom-Forming Populations and Expands Metabolite Structural Diversity

Séverine Le Manach et al. Front Microbiol.

Abstract

Cyanobacteria are photosynthetic prokaryotes capable of synthesizing a large variety of secondary metabolites that exhibit significant bioactivity or toxicity. Microcystis constitutes one of the most common cyanobacterial genera, forming the intensive blooms that nowadays arise in freshwater ecosystems worldwide. Species in this genus can produce numerous cyanotoxins (i.e., toxic cyanobacterial metabolites), which can be harmful to human health and aquatic organisms. To better understand variations in cyanotoxin production between clones of Microcystis species, we investigated the diversity of 24 strains isolated from the same blooms or from different populations in various geographical areas. Strains were compared by genotyping with 16S-ITS fragment sequencing and metabolite chemotyping using LC ESI-qTOF mass spectrometry. While genotyping can help to discriminate among different species, the global metabolome analysis revealed clearly discriminating molecular profiles among strains. These profiles could be clustered primarily according to their global metabolite content, then according to their genotype, and finally according to their sampling location. A global molecular network of all metabolites produced by Microcystis species highlights the production of a wide set of chemically diverse metabolites, including a few microcystins, many aeruginosins, microginins, cyanopeptolins, and anabaenopeptins, together with a large set of unknown molecules. These components, which constitute the molecular biodiversity of Microcystis species, still need to be investigated in terms of their structure and potential bioactivites (e.g., toxicity).

Keywords: aquatic environment; chemiodiversity; cyanobacteria blooms; mass spectrometry; secondary metabolites.

Figures

FIGURE 1
FIGURE 1
Microcystis spp. General view of a representative intense Microcystis sp. bloom in a recreational pound (Champs-sur-Marne, © B. Marie) (A). Macrograph of Microcystis colonies at surface water (© B. Marie) (B). Example of 15-mL vessels containing the monoclonal strains of Microcystis spp. maintained in the Paris’ Museum Collection (PMC) of cyanobacteria (MNHN, Paris, © C. Duval) (C). Example of micrograph of the isolated monoclonal culture of the Microcystis aeruginosa, where scale bare represents 10 μm (© C. Duval) (D). Representative picture of Microcystis aeruginosa cell from PMC 156.02 strain (here in division) under transmission electron microscope, where scale bare represents 0.5 μm (© C. Djediat) (E). General structures of various cyanobacterial metabolites belonging to the microcystin, anabaenopeptin, microginin, microviridin, aeruginosin and oscillatorin families (F).
FIGURE 2
FIGURE 2
Maximum likelihood (ML) tree based on partial 16S-23S ITS sequences. The sequences obtained in this study are indicated in bold. The strains, which exhibit mcyA gene, are indicated in red. Other sequences were retrieved from GenBank, accession numbers in brackets. Bootstrap values > 60% are shown at the nodes. The scale bar indicates number of nucleotide substitutions per site. The Microcystis sp. AICB832 was used as an out-group.
FIGURE 3
FIGURE 3
Heatmap representation of the metabolome of the 24 Microcystis spp. monoclonal strains analyzed using HR ESI-Qq-TOF, representing 2051 different analytes (in a 400–2000 Da window; present in at least three strains, with a signal to noise ratio in excess of 6, and respective relative peak intensity superior to 5000-count in at least one sample threshold) identified by MetaboScape software. The hierarchical clustering between strains was performed according to Bray-Curtis distance method. Green and blue squares indicate M. aeruginosa and M. wesenbergii/viridis, respectively. Sampling localities are: C, Champs-sur-Marne; B, Burkina Faso; E, Eure et Loire; Ne, Netherlands; Sc, Scotland; Se, Senegal; So, Souppes-sur-Loin; Var, Varennes-sur-Seine; Val, Valence; Vi, Villerest.
FIGURE 4
FIGURE 4
Molecular network generated from MS/MS spectra from the 24 Microcystis strains using the GNPS tool (all data and results are freely available at the address http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c017414365e84334b38ae75728715552). The nodes of the analytes detected in M. aeruginosa or M. wessenbergii/viridis strains only are indicated in green and blue, respectively, when analytes detected in both species are indicated in orange. Uncharacterized analytes are indicated by circles constituting potential new analogs. Analytes whom individual masses match with known secondary metabolites from cyanobacteria (listed in Supplementary Table S1) are indicated by specific shapes as shown on picture caption. Standard molecules analyses similarly, are indicated by heavy black lines. Only cluster regrouping at least 2 analytes are represented. A, di- or tri-peptide cluster; B, Unknown metabolite clusters; C, microcystin clusters; D, anabaenopeptin clusters; E, aeruginosin clusters; F, aerucyclamide clusters; G, microginin clusters; H, cyanopeptolin clusters. The clusters on the right are corresponding to un-annotated and smaller clusters.
FIGURE 5
FIGURE 5
Microcystin clusters (A–C) highlighted by the GNPS analysis based on the MS/MS CID fragmentation spectra obtained from the 24 Microcystis strains. Analytes detected in M. aeruginosa or M. wessenbergii/viridis strains only are indicated in green and blue, respectively, when analytes detected in both species are in orange. Analytes whom individual masses match with known microcystins (MCs) are indicated by hexagons. Standard molecules analyses similarly, are indicated by heavy black lines. Example of MS/MS spectra and chemical structures are shown for (Asp3)-microcystin-LR (D), microcystin-YR (E), -LR (F), -LF (G), -LA (H) and -HtyR (I). Notice that (M+H)+ and (M+2H)2+ ions may be grouped in distinct clusters (A–C, respectively).
FIGURE 6
FIGURE 6
Aeruginosin clusters (A,B) highlighted by the GNPS analysis based on the MS/MS CID fragmentation spectra obtained from the 24 Microcystis strains. Analytes detected in M. aeruginosa or M. wessenbergii/viridis strains only are indicated in green and blue, respectively, when analytes detected in both species are in orange. Analytes whom individual masses match with known aeruginosins are indicated by squares with right corners. Standard molecules analyses similarly, are indicated by heavy black lines. Example of MS/MS spectra and chemical structures are shown for aeruginosin 98A (C) and 98B (D).
FIGURE 7
FIGURE 7
Anabaenopeptin clusters (A–C) highlighted by the GNPS analysis based on the MS/MS CID fragmentation spectra obtained from the 24 Microcystis strains. Analytes detected in M. aeruginosa strains are indicated in green. Analytes whom individual masses match with known anabaenopeptins are indicated by squares with rounded corners. Standard molecules are indicated by heavy black lines. Uncharacterized analytes are indicated by circles constituting potential new analogs. Example of MS/MS spectra and chemical structures are shown for Oscillamide Y (D), anabaenopeptin F (E), A (F) and B (G). Notice that (M+H)+ and (M+2H)2+ ions may be represented by different nodes grouped in distinct clusters (A–C, respectively).
FIGURE 8
FIGURE 8
Cyanopeptolin clusters (A–F) highlighted by the GNPS analysis based on the MS/MS CID fragmentation spectra obtained from the 24 Microcystis strains. Analytes detected in M. aeruginosa strains are indicated in green. Analytes whom individual masses match with known cyanopeptolin are indicated by parallelepipeds. Standard molecules are indicated by heavy black lines. Uncharacterized analytes analyses similarly, are indicated by circles constituting potential new analogs. Example of MS/MS spectra and chemical structures are shown for cyanopeptolin B (G) and A (H).
FIGURE 9
FIGURE 9
Microginin clusters (A,B) highlighted by the GNPS analysis based on the MS/MS CID fragmentation spectra obtained from the 24 Microcystis strains. Analytes detected in M. aeruginosa strains only are indicated in green and blue, when analytes detected in both species are in orange. Analytes whom individual masses match with known microginin are indicated by 45°-tilted squares. Standard molecules analyses similarly, are indicated by heavy black lines. Uncharacterized analytes are indicated by circles constituting potential new analogs. Example of MS/MS spectra and chemical structures are shown for microginin FR2 (C), FR1 (D), 711 (E) and 757 (F). Notice that spectra of microginin BN578 and SD755 (are not shown here).
FIGURE 10
FIGURE 10
Aerucyclamide clusters (A–C) highlighted by the GNPS analysis based on the MS/MS CID fragmentation spectra obtained from the 24 Microcystis strains. Analytes detected in M. aeruginosa or M. wessenbergii/viridis strains only are indicated in green and blue, respectively, when analytes detected in both species are in orange. Analytes whom individual masses match with known aerucyclamides are indicated by octagons. Uncharacterized analytes are indicated by circles constituting potential new analogs. Standard molecules analyses similarly, are indicated by heavy black lines. Example of MS/MS spectra and chemical structures are shown for aerucyclamide A (D), B (E) and D (F).

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