While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria. IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.

Sustainable and effective water management plans must have a reliable risk assessment strategies for harmful cyanobacterial blooms (HABs) that would enable timely decisions to be made, thus avoiding the trespassing of ecological thresholds, leading to the collapse of ecosystem structure and function. Such strategies are usually based on cyanobacterial biomass and/or on the monitoring of known toxins, which may, however, in many cases, under- or over-represent the actual toxicity of the HAB. Therefore, in this study, by the application of growth-inhibition assays using different bacteria, algae, zooplankton and fish species, we assessed the toxicological potential of two cyanobacterial blooms that differed in total cyanobacterial biomass, species composition and cyanopeptide profiles. We demonstrated that neither cyanobacterial community composition nor its relative abundance, nor indeed concentrations of known toxins reflected the potential risk of HAB based on growth-inhibition assays. We discuss our findings in the context of food-web dynamics and ecosystem management, and suggest that toxicological tests should constitute a key element in the routine monitoring of water bodies so as to prevent under-/over-estimation of potential HAB risk for both ecosystem and public health.

Summary: Freshwater bloom‐forming cyanobacteria densely grow in the aquatic environments, leading to an increase in the viral‐contact rate. They possess numerous antiviral genes, as well as cell differentiation‐ and physiological performance‐related genes, owing to genome expansion. Their genomic features and unique lifestyles suggest that they coexist with cyanoviruses in ways different from marine cyanobacteria. Furthermore, genome contents of isolated freshwater bloom‐forming cyanobacterial viruses have little in common with those of marine cyanoviruses studied to date. They lack the marine cyanoviral hallmark genes that sustain photosynthetic activity and redirect host metabolism to viral reproduction; therefore, they are predicted to share metabolisms and precursor pools with host cyanobacteria to ensure efficient viral reproduction and avoid nutrient deficiencies and antiviral response. Additionally, cyanovirus–cyanobacteria coexistence strategies may change as bloom density increases. Diverse genotypic populations of cyanoviruses and hosts coexist and fluctuate under high viral‐contact rate conditions, leading to their rapid coevolution through antiviral responses. The ancestral and newly evolved genotypes coexist, thereby expanding the diversity levels of host and viral populations. Bottleneck events occurring due to season‐related decreases in bloom‐forming species abundance provide each genotype within cyanobacterial population an equal chance to increase in prevalence during the next bloom and enhance further diversification.

Cyanobacteria play a significant role in ecosystem functioning as photosynthetic and CO₂ fixing microorganisms. Whether and to what extent cyanophages alter these carbon and energy cycles in their cyanobacterial hosts is still poorly understood. In this study, we investigated changes in photosynthetic activity (PSII), expression of genes associated with the light phase of photosynthesis (psbA, petA, ndhK) and carbon metabolism (rbcL, zwf) as well as intracellular ATP and NADHP concentrations in freshwater bloom-forming filamentous cyanobacterium Aphanizomenon flos-aquae infected by cyanophage vB_AphaS-CL131. We found that PSII activity and expression level of rbcL genes, indicating potential for CO₂ fixation, had decreased in response to cyanophage adsorption and DNA injection. During the period of viral DNA replication and assembly, PSII performance and gene expression remained at this decreased level and did not change significantly, indicating lack of transcriptional shutdown by the cyanophage. Combined, these observations suggest that although there is little to no interference between cyanophage DNA replication, host transcription and cellular metabolism, A. flos-aquae underwent a physiological state-shift toward lower efficiency of carbon and energy cycling. This further suggest potential cascading effect for co-occurring non-infected members of the microbial community.