Cyanotoxins called microcystins (MCs) are highly toxic and can be present in drinking water sources. Determining the structure of MCs is paramount because of its effect on toxicity. Though over 300 MC congeners have been discovered, many remain unidentified. Herein, a method is described for the putative identification of MCs using liquid chromatography (LC) coupled with high-resolution (HR) Orbitrap mass spectrometry (MS) and a new bottom-up sequencing strategy. Maumee River water samples were collected during a harmful algal bloom and analyzed by LC−MS with simultaneous HRMS and MS/ MS. Unidentified ions with characteristic MC fragments (135 and 213 m/z) were recognized as possible novel MC congeners. An innovative workflow was developed for the putative identification of these ions. Python code was written to generate the potential structures of unidentified MCs and to assign ions after the fragmentation for structural confirmation. The workflow enabled the putative identification of eight previously reported MCs for which standards are not available and two newly discovered congeners, MC-HarR and MC-E(OMe)R.

Cyanobacteria harmful algal blooms (cyanoHABs) dominated byMicrocystisspp. have significant public health and economic implications in freshwater bodies around the world. These blooms are capable of producing a variety of cyanotoxins, including microcystins, that affect fishing and tourism industries, human and environmental health, and access to drinking water. In this study, we isolated and sequenced the genomes of 21 unialgalMicrocystiscultures collected from western Lake Erie between 2017-2019. While some cultures isolated in different years have a high degree of genetic similarity (Average Nucleotide Identity >99%), genomic data shows that these cultures also represent much of the breadth of knownMicrocystisdiversity in natural populations. Only 5 isolates contained all the genes required for microcystin synthesis while 2 isolates contained a previously described partialmcyoperon. Microcystin production within cultures was also assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and supported genomic results with high concentrations (up to 900 μg L-1) in cultures with completemcyoperons and no or low toxin detected otherwise. These xenic cultures also contained a substantial diversity of bacteria associated withMicrocystis, which has become increasingly recognized as an essential component of cyanoHAB community dynamics. These results highlight the genomic diversity amongMicrocystisstrains and associated bacteria in Lake Erie, and their potential impacts on bloom development, toxin production, and toxin degradation. This collection significantly increases the availability of environmentally relevantMicrocystisstrains from temperate North America, which is changing rapidly due to climate change.HighlightsTwenty one xenicMicrocystiscultures were isolated from western Lake Erie and capture the diversity ofMicrocystisstrains observed in natural populations as well as their associated bacteriaMicrocystisstrains show variability in core and accessory gene content, and genetically similar strains produce varying concentrations and congeners of microcystinsThis collection is a valuable resource for studying strain diversity and interactions betweenMicrocystisand associated bacteriaOur collection increases the availability of environmentally relevant strains from temperate North America, which is historically underrepresented in culture collections.

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