Genomics of Harmful Freshwater Cyanobacteria – Microcystis

Microcystis, a cyanobacteria genus, frequently causes harmful algal blooms in freshwater lakes. Researching its genome helps understand its environmental adaptability and informs strategies to mitigate its detrimental impacts.

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Table of Contents

Key Takeaways
  • The toxin-producing freshwater Cyanobacteria – Microcystis were found in the lakes surrounding the Frederiksborg Castle in Denmark during the summer time of 2023.

  • Fourteen complete Microcystis genomes were analyzed with various genomics tools.

  • Over 50% of the Microcystis pangenome consists of accessory or unique genes, suggesting a trade-off between gene gains and losses in their evolutionary pattern.

Microcystis Cells
What is Microcystis?
  1. Cyanobacteria: Microcystis belongs to the group of cyanobacteria, conventionally called blue-green algae, organisms that are important in aquatic ecosystems due to their ability to perform photosynthesis. They contribute to primary production in these ecosystems.

  2. Morphology: Microcystis species typically form colonies that are composed of cells surrounded by mucilaginous sheaths. This characteristic structure can create a scum-like appearance on the surface of water bodies when they bloom.

  3. Harmful Algal Blooms (HABs): One of the most notable features of Microcystis is its tendency to form harmful algal blooms. These blooms can produce potent toxins, most notably microcystins, which are harmful to wildlife, domestic animals, and humans.

Why Research Microcystis?
  1. Public Health and Safety: Understanding Microcystis’ genomics is crucial for predicting and managing the risks posed by its toxins, which can contaminate drinking and recreational waters, leading to serious health hazards.

  2. Environmental Protection and Ecosystem Balance: Genomic research aids in comprehending and mitigating the negative impacts of Microcystis blooms on aquatic ecosystems, such as oxygen depletion, sunlight blockage, and disruption of food web dynamics. It also supports effective ecosystem monitoring and management.

  3. Scientific and Biotechnological Advancements: Studying Microcystis’ genetic makeup provides insights into their adaptation mechanisms, particularly in response to climate change, and unveils opportunities in biotechnology, like biofuel production and bioremediation, by understanding the mechanisms of toxin production and potential biotechnological applications.

Study Design
Microcystis in the Frederiksborg Castle Lake

The Slotssøen Lake is covered with a thick, greenish layer caused by a bloom of Microcystis, a type of blue-green algae. This greenish layer floats on the surface and often gathers around the edges or spreads across the water, moved by wind and currents. This not only changes how the lake looks, making it less clear and more like a green soup, but also harms the water’s health. The algae block sunlight from reaching deeper into the water, affecting other aquatic life, and can produce toxins that are dangerous to people, animals, and the environment

Sampling of Microcystis Biomass
1. Field Collection at the Lake
  • Preparation: Wear sterile gloves.
  • Sampling: Use a 50 ml centrifuge tube to collect floating algae from the lake’s surface. Ensure the sample volume does not exceed 50% of the tube’s capacity to avoid creating an anaerobic environment due to high biomass and limited air volume.
  • Settling: Allow the sample to settle for about 5 minutes. A qualified sample should have a 0.5 to 1.0 cm thick layer of algae on top. If the layer is too thin, collect additional samples from different locations in the lake.
  • Storage: Immediately store the sample in a foam box with ice packs.
2. Laboratory Processing
  • Settling: Allow the 50 ml centrifuge tube to stand still for 10 to 30 minutes, so all particles float to the top.
3. Preparing for Microscopic Examination
  • Setup: Prepare three Petri dishes and place them in order. Add 3 to 5 ml of sterile water to each dish.
4. Algae Transfer and Washing
  • Transfer Tool Preparation: Use a 1 ml pipette tip, cutting off the tip end to facilitate the transfer of larger volumes.
  • Algae Transfer: Transfer approximately 50 μl (volume adjusted based on cyanobacteria concentration) of algae from the 50 ml tube to the first Petri dish.
  • Washing Procedure:
    • In the first dish, the algae particles will disperse and float. Use a sterile inoculating loop to pick the largest particles and transfer them to the second dish for rinsing.
    • Repeat the transfer to the third dish. This step aims to wash off loosely adhering bacteria from the algae particles.
  • Final Transfer: Transfer the washed algae particle into a 1.5 ml Eppendorf tube or a 2 ml cryotube. Add 0.2 ml of TE buffer to ensure DNA preservation.
DNA Extraction, Sequencing and Bioinformatics

DNA extraction and sequencing

Microbiome DNA was extracted using a FastPure Microbiome DNA Isolation Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. DNA quantity and quality were assessed with a nano-spectrophotometer at mBioWorks Copenhagen, Denmark. The sequencing library was constructed according to standard DNA nanoballs (DNB)-based protocols from MGI. The DNA library was paired-end sequenced (2×150) on an MGISEQ-2000 sequencer.

Bioinformatic Pipeline

  • Adapter/quality trimming: fastp
  • Assembly: spades/unicycler
  • Assembly report: quast
  • Genome classification: gtdbtk
  • Quality assessment: checkm
  • Genome annotation: pgap, DRAMs, RAST
  • Pangenome: Anvio
  • Secondary metabolites: Antismash
  • Ancestor reconstruction: FastDTLmapper

The following Microcystis genomes were included in the following data visualization.

Raw reads quality report

A typical read quality report generated with fastqc or fastp:

Before filtering of reads:

After filtering of reads:

Genome assembly assessment
Genome annotation
Identification of secondary metabolites biosynthesis gene cluster
Reconstruction of energy/metabolism pathways

Sulfur cycle as an example.


Phylogenomic tree was depicted to reveal the phylogenetic relationships within the genus Microcystis, based on 92 Core Gene Sequences. The numbers at each node indicate gene support indexes, with the maximum value being 92. Ma_S464 is used as the outgroup for this analysis. The scale bar denotes 0.001 substitutions per nucleotide position.

Pangenome of Microcystis

This figure illustrates the gene clusters arranged according to their prevalence across different Microcystis genomes, with clusters shared by the same group of isolates positioned nearer to each other. The genomes are organized based on their Average Nucleotide Identity (ANI) values. The subsequent two layers provide details on gene clusters where at least one gene has been functionally annotated, either through Pfam or COG databases.

The pan-genome analysis of Microcystis reveals significant intra- and inter-cluster genetic variations. A notable observation is the identical pan-genomes within specific clusters. For example, in Cluster 2, the genomes Ma_NIES2481 and Ma_NIES2549 exhibit identical pan-genomes. Similarly, Ma_PCC7806, Ma_PCC7806SC, and Ma_DIANCHI905 within Cluster 1 also share identical pan-genomes.

Ancestral reconstruction of Microcystis

The Reconstruction of the Last Common Ancestor of Microcystis (N002) and Its Genomic Features: This ancestor was reconstructed with approximately 4,346 protein-coding genes. Out of these, 1,595 genes were identified as gains, many of which are linked to DNA editing processes, including 108 genes associated with functions such as transposase, endodeoxyribonuclease, and deoxyribonuclease activities. In the context of Microcystis aeruginosa PCC 7806, it has been noted that about 6.8% of the genes are putative transposases. The most frequently gained or lost genes in ancestral genomes pertain to DNA editing, transporters, and ion or acid binding functions (as detailed in Table 3). A comparative analysis reveals that Cluster 2, in contrast to Clusters 1 and 3, experienced a higher loss of genes than gains, resulting in smaller genome sizes. Notably, the three genomes within Cluster 2 are characterized as non-toxic strains.


The combination of 16S rRNA gene phylogenetic analysis and average nucleotide identity (ANI) supports the notion of Microcystis as a single species. Yet, this classification raises complexities in ecological studies. Our comprehensive pangenome analysis, encompassing 14 complete Microcystis genomes, uncovered that a significant portion—over 50%—of the pangenome is comprised of accessory or unique genes. This diversity within the pangenome highlights the genus’s genetic variability despite its apparent taxonomic singularity. Further, the ancestral reconstruction of gene content evolution in Microcystis reveals a dynamic interplay of gene gains and losses, particularly in areas related to DNA editing, transport, and ion or acid binding. These evolutionary trends reflect a balance between genome expansion and reduction, indicating a nuanced evolutionary strategy within the Microcystis genus. This strategy appears to be driven by the need to adapt to varied environmental conditions while maintaining core functional capabilities. Our findings underscore the value of advanced genomic analyses in unraveling the complex evolutionary pathways of ecologically significant cyanobacteria like Microcystis.

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