The Biosynthetic Repertoire of Microcystis spp. in Western Lake Erie Harmful Algal Blooms: Insights into Biosynthesis of Known and Novel Secondary Metabolites in Natural Populations using a `Multi-Omic' Approach

Abstract

Microcystis species threaten freshwaters around the world through the formation of cyanobacteria harmful algal blooms (cyanoHABs). These blooms can disrupt ecosystem function, negatively impact tourism and industry, and most notably, cause human and animal illness through the production of toxins. As climate change intensifies, cyanoHABs are expected to become more widespread and persistent. Due to its potency and general ubiquity, the hepatotoxin microcystin is of great concern; however, it is not the only secondary metabolite produced by Microcystis. Several Microcystis-derived secondary metabolites, or accessory chemical compounds that provide the producer with an advantage, have toxic properties, yet the full extent of their persistence, toxicity, and distribution remains unknown. Additionally, Microcystis harbor cryptic gene clusters thought to encode unknown secondary metabolites. Beyond threats to human and ecosystem health, these secondary metabolites may also have application in pharmaceutical discovery. To address knowledge gaps pertaining to Microcystis secondary metabolism, a multi-omics approach was used to analyze cultured isolates and natural cyanoHAB communities from Western Lake Erie. By using paired (meta)genomic, (meta)transcriptomic, and metabolomic datasets, cyanobacterial derived secondary metabolites were deeply explored to understand the breadth and diversity of biosynthetic gene clusters (BGCs), their patterns of expression and potential drivers from natural environments, as well as the chemical diversity of produced molecules. Overarching themes include the discovery and characterization of novel BGCs, putatively linking BGCs to produced metabolites, assessing shifts in BGC abundance and expression along environmental gradients, and investigating the diversity of BGC composition and structure across different Microcystis strains. Observations from western Lake Erie cyanoHABs reveal dynamic shifts in the relative abundance and expression of numerous BGCs with implications for secondary metabolite discovery and water quality. In Chapter 2, the mcy operon, which encodes microcystin biosynthesis, was explored and revealed a novel partial mcy operon, which is abundant and transcriptionally active through time, suggesting its ecological importance. In Chapter 3, several BGCs were detected in Microcystis populations that encode biosynthesis of numerous secondary metabolites. These BGCs putatively encode for compounds with toxic and/or taste and odor properties, as well as cryptic metabolites. Relationships between both abiotic and biotic variables and BGC transcription suggest multifaceted controls on biosynthesis in natural environments. In Chapter 7, less abundant cyanobacteria from the Order Nostocales were examined for biosynthetic potential, revealing that other cyanobacteria can contribute to the secondary metabolite pool, although BGCs encoding known neurotoxins were not detected during the time of sampling. Through cultivation studies, greater insights into biosynthetic pathways and synthesis products were possible. In Chapter 6, tetrapeptides hypothesized to be the synthesis products of partial mcy operons, were detected only in Microcystis isolates with the partial genotype supporting the conclusion that partial operons encode biosynthesis products. In Chapters 4 and 5, the Western Lake Erie Culture Collection provided an opportunity for deep analysis of biosynthesis pathways encoded in Microcystis genomes and the chemical diversity observed within the metabolome. This work highlights that the vast majority of the Microcystis metabolome remains poorly understood, and is distinct across different, but closely related, strains. The extensive genetic and chemical diversity observed within isolates also emphasizes the ability of Microcystis to biosynthesize several known molecules with toxic properties. Together, these results underscore the ability of Microcystis to produce a diverse range of secondary metabolites that have implications for human and environmental health.