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. 2024 Jul 23;9(7):e0033424.
doi: 10.1128/msystems.00334-24. Epub 2024 Jun 25.

Metabologenomics reveals strain-level genetic and chemical diversity of Microcystis secondary metabolism

Colleen E Yancey  1 , Lauren Hart  2   3 , Sierra Hefferan  2   4 , Osama G Mohamed  3   5   6 , Sean A Newmister  3 , Ashootosh Tripathi  3   5   6 , David H Sherman  3   4   6 , Gregory J Dick  1   2   7
Affiliations

Metabologenomics reveals strain-level genetic and chemical diversity of Microcystis secondary metabolism

Colleen E Yancey et al. mSystems. .

Abstract

Microcystis spp. are renowned for producing the hepatotoxin microcystin in freshwater cyanobacterial harmful algal blooms around the world, threatening drinking water supplies and public and environmental health. However, Microcystis genomes also harbor numerous biosynthetic gene clusters (BGCs) encoding the biosynthesis of other secondary metabolites, including many with toxic properties. Most of these BGCs are uncharacterized and currently lack links to biosynthesis products. However, recent field studies show that many of these BGCs are abundant and transcriptionally active in natural communities, suggesting potentially important yet unknown roles in bloom ecology and water quality. Here, we analyzed 21 xenic Microcystis cultures isolated from western Lake Erie to investigate the diversity of the biosynthetic potential of this genus. Through metabologenomic and in silico approaches, we show that these Microcystis strains contain variable BGCs, previously observed in natural populations, and encode distinct metabolomes across cultures. Additionally, we find that the majority of metabolites and gene clusters are uncharacterized, highlighting our limited understanding of the chemical repertoire of Microcystis spp. Due to the complex metabolomes observed in culture, which contain a wealth of diverse congeners as well as unknown metabolites, these results underscore the need to deeply explore and identify secondary metabolites produced by Microcystis beyond microcystins to assess their impacts on human and environmental health.IMPORTANCEThe genus Microcystis forms dense cyanobacterial harmful algal blooms (cyanoHABs) and can produce the toxin microcystin, which has been responsible for drinking water crises around the world. While microcystins are of great concern, Microcystis also produces an abundance of other secondary metabolites that may be of interest due to their potential for toxicity, ecological importance, or pharmaceutical applications. In this study, we combine genomic and metabolomic approaches to study the genes responsible for the biosynthesis of secondary metabolites as well as the chemical diversity of produced metabolites in Microcystis strains from the Western Lake Erie Culture Collection. This unique collection comprises Microcystis strains that were directly isolated from western Lake Erie, which experiences substantial cyanoHAB events annually and has had negative impacts on drinking water, tourism, and industry.

Keywords: Microcystis; cyanoHABs; metabologenomics; natural products; secondary metabolism.

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Conflict of interest statement

C.E.Y. was a graduate student at the time this research was completed but is now an employee at New England Biolabs, Ipswich, MA, USA.

Figures

Fig 1
Fig 1
Overview of BGCs in WLE Microcystis culture isolates. A phylogenetic tree based on concatenated universal single-copy genes, of the Microcystis isolates in the WLECC, as previously described by Yancey et al. (43). Colored boxes indicate that the presence or absence of common Microcystis secondary metabolite genes includes the mcy (microcystin), aer (aeruginosin), mcn (cyanopeptolin/micropeptin), apn (anabaenopeptin), and mvd (microviridin B) operons, as well as the BGC, PmNT3, which may encode the biosynthesis of a paracyclophane molecule. The bar graph indicates the putative count and broad classification of BGCs within each Microcystis genome.
Fig 2
Fig 2
GCFs for Identified BGCs via BiG-SCAPE: Green nodes are from the WLECC, and yellow nodes are from the 2014 WLE cyanoHAB metagenomes. Nodes in purple are from the MiBIG database, which links BGCs with biosynthesis products. Edges between the nodes indicate the similarity of BGCs as calculated through a distance matrix that includes indexes that measure the number of shared PFAM domains, pairs of adjacent PFAM domains, and sequence similarities between predicted protein sequences.
Fig 3
Fig 3
Gene schematics, gene annotations, and putative-related biosynthetic products of cryptic BGC PmNT3. (A) Detailed gene schematic and generated GCF tree from antiSMASH and BiG-SCAPE, respectively. Key genes are annotated on the large gene schematic. (B) Known cluster BLAST generated via antiSMASH. The novel BGC shows 30%–55% similarity of gene content to known cyanobacterial BGCs encoding for the biosynthesis of [7, 7] paracyclophanes: merocyclophane C/D (55%), cylindrocyclophane (41%), and carbamidocyclophane (31%). (C) Chemical structures for select cyanobacterial [7, 7] paracyclophanes that are confirmed biosynthesis products from the order Nostacales.
Fig 4
Fig 4
Molecular network representing the WLECC metabolome from positive mode liquid chromatography-mass spectrometry (LC-MS). Nodes are colored based on feature frequency within WLECC cultures (ranging from being found in one culture to all analyzed cultures). Metabolites annotated by GNPS, DEREPLICATOR, and SNAP-MS are outlined in red, light blue, and gray, respectively. Several known Microcystis secondary metabolites were putatively identified including microcystins, anabaenopeptins, and features with similarity to micropeptins/cyanopeoptolins, microginins, and minutissamides.
Fig 5
Fig 5
BGC and chemical feature absence matrix for select secondary metabolites: Cultures are arranged based on the phylogenetic tree generated via concatenated single-copy housekeeping genes as described in Yancey et al. (40, 43). On the top matrix, colored boxes indicate the presence of known BGCs (mcy, aer, mcn, apn, and mgn gene clusters). The light purple coloring indicates the presence of the partial mcy operon. Circles indicate the presence of an annotated chemical feature from each culture. Black circles were annotated with GNPS (highest confidence), and gray circles were annotated with either DREPLICATOR or SNAP-MS. On the bottom matrix, colored boxes indicate the presence of cryptic BGCs (annotated in Fig. 2). Gray coloring indicates the presence of core biosynthesis within the PmNT3 cluster in LE19-196.1 White circles indicate the presence of the top-scoring candidate chemical feature with respect to each BGC (NRPS 1, NRPS 2, Hyb1, Hyb 2, and PmNT3), as determined by NPlinker. The Metcalf score and parent mass for top hits are depicted in the box to the right.
Fig 6
Fig 6
Cyanopeptolin/micropeptin molecular networking and gene cluster analysis. (A) The molecular network with micropeptin cluster was obtained from the WLECC with a cosine similarity score cutoff of 0.70. Nodes are labeled with corresponding cluster index numbers assigned through GNPS, and their m/z are shown in black text next to each pair of nodes that share the same m/z. Edges between nodes show the mass difference between sets of nodes. Two micropeptins with edges outlined in purple indicate GNPS probable structure detection through GNPS DEREPLICATOR. Pie charts within nodes indicate strains that the metabolites were detected in. Cluster index 843 corresponds to CyanoMetDB micropeptin MZ 845 shown in the figure with the experimental MS/MS [M + H]+ annotated data shown. Ahp is 3-amino-6-hydroxy-2-piperidone. Cluster index 1,269 had a predicted structure from GNPS DEREPLICATOR but does not correspond to experimental MS/MS data, and the structure remains unidentified. (B) Gene schematics for three gene clusters identified in three WLECC strains, and their comparison to known mcn gene clusters deposited in the Minimum Information about a Biosynthetic Gene Cluster (MiBIG) database (https://mibig.secondarymetabolites.org/repository). Genes are colored based on PFAM domain annotation for each cluster.
Fig 7
Fig 7
Spectra linked to the anabaenopeptin (A) apn and (B) PmNT3 gene clusters through NPLinker. Linked spectral features identified by NPlinker are colored orange, and the parent mass, Metcalf score, number of shared strains, and putative annotation via GNPS and/or ClassyFire are reported in the table. Gray nodes represent features that were not linked with NPlinker but were within subnetworks for putatively linked features. For the PmNT3 cluster, select molecular fingerprints including ring systems, halogenations, or sulfurous qualities are highlighted with differing colors.
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