Investigating Cyanobacterial Communities Across Canada and Characterizing the Seasonality of Cyanobacterial Toxin and Nitrogen Metabolism Genes in the Turkey Lakes Watershed Using Metagenomics

The long evolutionary history of gram-negative cyanobacteria has enabled them to tolerate extreme conditions, succeed in numerous habitats, and exploit special nutrients, all while resisting harmful anthropogenic and climate change exacerbated influences. Increases in harmful cyanobacterial blooms (cHABs) pose considerable challenges to the provision of safe drinking water globally. Cyanobacterial blooms are most often recorded in eutrophic lakes where they have sufficient nutrients for proliferation, however, this has led to the misunderstanding that they do not pose risks in oligotrophic lakes because of the lower nutrient availability needed to form harmful blooms. Yet, the absence of visual surface blooms does not correlate to the absence of cyanobacteria as they can be abundant at different depths within the water column. Cyanobacteria may release toxic metabolites into water sources and if left untreated, these toxins threaten drinking water security, including those from oligotrophic lakes, hence, the high demand for research in drinking water treatability sectors and sustainable forested watershed management. The distribution of cyanobacterial communities across Canada was investigated in this study (Chapter 2); samples were collected by various forWater HQP from five major forested ecozones: Atlantic and Pacific Maritime, Boreal Shield and Plains, and the Montane Cordillera. Unique cyanobacterial ASVs obtained from V4-16S rRNA region gene sequencing of all samples were phylogenetically analysed using Maximum Likelihood tree models to observe cyanobacterial taxonomy and biogeography within the various research platforms. Understanding the composition of cyanobacterial communities is a valuable tool in the proactive monitoring of threats to water systems because by observing microbial communities, it can help identify major toxin producing species and illustrate the overall quality of the source water. This research demonstrated that cyanobacteria across Canada are highly diverse and spread across the country, cyanobacteria are unaffected by geographical restrictions as the same taxa can be observed in any of the five ecozones, and there is a significant gap in sequenced picocyanobacteria (0.2-2 μm) in genomic libraries. Whole genome shotgun metagenomic sequencing uses untargeted sequencing to characterize the functional gene diversity and biochemical potential of entire environmental DNA microbial communities. Its application is a valuable tool in observing the potential for toxin synthesis and taste/odour compounds that may negatively impact the state of drinking/recreational water systems. As the only toxin regulated in Canada (1.5 μg/L drinking water, 10 μg/L recreational waters), microcystin damages the liver and kidneys by acting as hepatocytes and disrupting intracellular protein regulation. Lipid A is the toxic component of lipopolysaccharides (endotoxins produced by gram-negative bacteria) which initiate dangerous immune responses and is the culprit for most gram-negative diseases. Alternatively, nitrogen- fixation by cyanobacteria converts atmospheric nitrogen into usable intracellular N2 supplies, allowing for optimal nitrogen availability that is crucial for proliferation and gene expression. The potential for organisms to produce such toxins or be capable of fixing nitrogen is dependent on the presence of biosynthetic gene clusters encoding the necessary genes. Several gene markers were targeted that represent the presence of potential biosynthesis of toxins and N2-metabolic abilities. This research (Chapter 3) characterized seasonal distribution of two toxin gene families: microcystin (mcy) and lipoxygenase (lpx), and three nitrogen metabolism genes families: cyanase (cynS), nitrogenase (nif), and a global nitrogen regulator (ntcA). Genes within the biosynthetic mcy gene cluster signify the production of microcystin, and lpx genes encode for lipid A endotoxins; the cynS gene is involved in fixing cyanate (an organic nitrogen source), the nif gene cluster encodes the primary nitrogen fixation enzyme family, nitrogenase, and ntcA is a global nitrogen regulator crucial for all nitrogen assimilation and acquisition within cyanobacteria. The target toxin genes (mcy, lpx) were chosen for their potential to harm individuals infected with contaminated water, and the target nitrogen-metabolism genes (cynS, nif, ntcA) were chosen due to their capability of providing nitrogen to cyanobacteria which allows them to proliferate, thrive, and express genes even when nitrogen levels in the water are not optimal. The target gene abundances were observed over six sampling points, including under an ice-covered period, using metagenomic analysis, to identify if there is a link among seasonal gene observations.

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