Metagenomic and Metatranscriptomic Insights into Population Diversity of Microcystis Blooms: Spatial and Temporal Dynamics of mcy Genotypes, Including a Partial Operon That Can Be Abundant and Expressed

Abstract

Cyanobacterial harmful algal blooms (cyanoHABs) degrade freshwater ecosystems globally. Microcystis aeruginosa often dominates cyanoHABs and produces microcystin (MC), a class of hepatotoxins that poses threats to human and animal health. Microcystin toxicity is influenced by distinct structural elements across a diversity of related molecules encoded by variant mcy operons. However, the composition and distribution of mcy operon variants in natural blooms remain poorly understood. Here, we characterized the variant composition of mcy genes in western Lake Erie Microcystis blooms from 2014 and 2018. Sampling was conducted across several spatial and temporal scales, including different bloom phases within 2014, extensive spatial coverage on the same day (2018), and frequent, autonomous sampling over a 2-week period (2018). Mapping of metagenomic and metatranscriptomic sequences to reference sequences revealed three Microcystis mcy genotypes: complete (all genes present [mcyA-J]), partial (truncated mcyA, complete mcyBC, and missing mcyD-J), and absent (no mcy genes). We also detected two different variants of mcyB that may influence the production of microcystin congeners. The relative abundance of these genotypes was correlated with pH and nitrate concentrations. Metatranscriptomic analysis revealed that partial operons were, at times, the most abundant genotype and expressed in situ, suggesting the potential biosynthesis of truncated products. Quantification of genetic divergence between genotypes suggests that the observed strains are the result of preexisting heterogeneity rather than de novo mutation during the sampling period. Overall, our results show that natural Microcystis populations contain several cooccurring mcy genotypes that dynamically shift in abundance spatiotemporally via strain succession and likely influence the observed diversity of the produced congeners. IMPORTANCE Cyanobacteria are responsible for producing microcystins (MCs), a class of potent and structurally diverse toxins, in freshwater systems around the world. While microcystins have been studied for over 50 years, the diversity of their chemical forms and how this variation is encoded at the genetic level remain poorly understood, especially within natural populations of cyanobacterial harmful algal blooms (cyanoHABs). Here, we leverage community DNA and RNA sequences to track shifts in mcy genes responsible for producing microcystin, uncovering the relative abundance, expression, and variation of these genes. We studied this phenomenon in western Lake Erie, which suffers annually from cyanoHAB events, with impacts on drinking water, recreation, tourism, and commercial fishing.

Keywords: Lake Erie; Microcystis; freshwater harmful algal blooms; mcy operon; metagenomics.

FIG 1

Metagenomic read mapping suggests three distinct mcy genotypes in western Lake Erie Microcystis populations at different times and locations. Genotypes were inferred by mapping metagenomic reads onto the reference mcy operon from Microcystis aeruginosa reference strain PCC 7806 (GenBank accession number AF183408.1). For each plot, the top panel indicates the percent identity of the read mapped to the reference operon, and the bottom panel shows coverage (number of reads mapped per position). The color of the genes indicates modular enzymes encoded by mcy genes. (A) Mapping results from a sample dominated by the complete genotype (WE12, 4 August). The asterisks indicate evidence of recombination in which the C1 domain replaced the B1 domain, creating a C1-like mcyB genotype, which is interpreted to dominate the metagenome of this sample (bottom operon). The lack of this recombination preserves the mcyB orientation for a B1-like mcyB genotype (top operon), as in the reference sequence from PCC 7806. (B) Sample dominated by the partial genotype (WE12, 23 September). (C) Absent genotype with no mcy genes present.

FIG 2

Relative abundances of Microcystis mcy genotypes from the 2014 cyanoHAB by station and date. Relative abundances of genotypes were calculated by taking the ratio of the coverage of mcy genes to the coverage of the V4 16S rRNA gene. (A) Proportion of the Microcystis population that contained a complete, partial, or absent mcy operon by sample. Values above 1 indicate higher coverage of mcy genes than the 16S rRNA genes (see Materials and Methods). (B) Proportion of the Microcystis population that contains either the mcyB C1-like or B1-like variant. (C) Concentrations of particulate microcystins (MCs), nitrate (NO₃⁻), and phycocyanin (PC) per sample (micrograms per liter).

FIG 3

Relative abundances of mcy genotypes for Microcystis populations sampled during the 2018 HABs Grab and 2018 ESP monitoring efforts. Relative abundances were calculated by taking the ratio of the coverage of mcy genes to the coverage of the V4 16S rRNA gene. The x axis is the distance from the Maumee River (kilometers) from which the sample was collected. (A) Proportion of the population containing a complete, partial, or absent mcy operon. (B) Proportion of the population containing the C1-like or B1-like mcyB variant for both the 2018 HABs Grab and 3G-ESP sampling efforts.

FIG 4

Hierarchical clustering of 2014 metagenomic samples based on pairwise conANI comparisons. Clustering indicates that the Microcystis strain composition is more similar in samples from the same bloom phase than in those from different phases that tend to have the same dominant genotype. The bloom phase described as “middle” in the illustration above refers to samples collected in August and September where the Microcystis biomass and microcystin concentration are still detectable yet past peak concentrations.

FIG 5

Heatmaps showing the relative abundances of metatranscriptomic reads mapped to the mcy operon. Relative abundance is determined by calculating the coverage of the gene of interest (number of transcript reads mapped divided by gene length) divided by the coverage of transcripts mapped to the entire genome divided by genome length.

FIG 6

Map of western Lake Erie HAB long-term monitoring stations, 2018 HABs Grab sample locations, and 2018 3G-ESP sample collection points. Western Lake Erie long-term monitoring stations are denoted with large red circles, 2018 HABs Grab samples are green circles, and 3G-ESP sample locations are orange circles. (The Open Street Map [OSM] [https://wiki.osmfoundation.org/wiki/Main_Page] was used as the basis for the map.)