Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions.

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.

Cyanobacteria and Their Metabolites in Mono- and Polidominant Shallow Eutrophic Temperate Lakes.

Monodominant (one species dominates) or polidominant (multiple species dominate) cyanobacterial blooms are pronounced in productive freshwater ecosystems and pose a potential threat to the biota due to the synthesis of toxins. Seasonal changes in cyanobacteria species and cyanometabolites composition were studied in two shallow temperate eutrophic lakes. Data on cyanobacteria biomass and diversity of dominant species in the lakes were combined with chemical and molecular analyses of fifteen potentially toxin-producing cyanobacteria species (248 isolates from the lakes). Anatoxin-a, saxitoxin, microcystins and other non-ribosomal peptides formed the diverse profiles in monodominant (Planktothrix agardhii) and polidominant (Aphanizomenon gracile, Limnothrix spp. and Planktolyngbya limnetica) lakes. However, the harmfulness of the blooms depended on the ability of the dominant species to synthesize cyanometabolites. It was confirmed that P. agardhii produced a greater amount and diverse range of MCs and other NRPs. In the polidominant lake, isolates of the co-dominant A. gracile, L. planctonica and P. limnetica synthesized no or only small amounts of cyanometabolites. In general, the profile of cyanometabolites was greater in cyanobacteria isolates than in environmental samples, indicating a high potential for toxic cyanobacteria bloom.

Exploring Viral Diversity in a Gypsum Karst Lake Ecosystem Using Targeted Single-Cell Genomics.

Little is known about the diversity and distribution of viruses infecting green sulfur bacteria (GSB) thriving in euxinic (sulfuric and anoxic) habitats, including gypsum karst lake ecosystems. In this study, we used targeted cell sorting combined with single-cell sequencing to gain insights into the gene content and genomic potential of viruses infecting sulfur-oxidizing bacteria Chlorobium clathratiforme, obtained from water samples collected during summer stratification in gypsum karst Lake Kirkilai (Lithuania). In total, 82 viral contigs were bioinformatically identified in 62 single amplified genomes (SAGs) of C. clathratiforme. The majority of viral gene and protein sequences showed little to no similarity with phage sequences in public databases, uncovering the vast diversity of previously undescribed GSB viruses. We observed a high level of lysogenization in the C. clathratiforme population, as 87% SAGs contained intact prophages. Among the thirty identified auxiliary metabolic genes (AMGs), two, thiosulfate sulfurtransferase (TST) and thioredoxin-dependent phosphoadenosine phosphosulfate (PAPS) reductase (cysH), were found to be involved in the oxidation of inorganic sulfur compounds, suggesting that viruses can influence the metabolism and cycling of this essential element. Finally, the analysis of CRISPR spacers retrieved from the consensus C. clathratiforme genome imply persistent and active virus-host interactions for several putative phages prevalent among C. clathratiforme SAGs. Overall, this study provides a glimpse into the diversity of phages associated with naturally occurring and highly abundant sulfur-oxidizing bacteria.

Interplay of Nutrients, Temperature, and Competition of Native and Alien Cyanobacteria Species Growth and Cyanotoxin Production in Temperate Lakes.

Global warming and eutrophication contribute to formation of HABs and distribution of alien cyanobacteria northward. The current study assessed how alien to Europe Sphaerospermopsis aphanizomenoides and Chrysosporum bergii will co-occur with dominant native Planktothrix agardhii and Aphanizomenon gracile species under changing conditions in temperate freshwaters. The experiments were carried out to examine the effect of nutrients and temperature on the growth rate of cyanobacteria, production of cyanotoxins, and interspecies competition. The highest growth rate was determined for A. gracile (0.43 day-1) and S. aphanizomenoides (0.40 day-1) strains at all the tested nutrient concentrations (IP and IN were significant factors). S. aphanizomenoides adapted to the wide range of nutrient concentrations and temperature due to high species ecological plasticity; however, A. gracile was able to suppress its dominance under changing conditions. Regularity between tested variables and STX concentration in A. gracile was not found, but IP concentration negatively correlated with the amount of dmMC-RR and other non-ribosomal peptides (NRPs) in P. agardhii strains. The relative concentration of NRPs in nontoxic P. agardhii strain was up to 3-fold higher than in MC-producing strain. Our study indicated that nutrients, temperature, and species had significant effects on interspecies competition. A. gracile had a negative effect on biomass of both alien species and P. agardhii.

Baltic Sea water tritium and stable isotopes in 2016-2017.

Water samples from the different basins of the Baltic Sea and North Sea were collected during four cruises taken place in 2016-2017. Altogether 60 water samples for 3H activity concentration and 120 water samples for stable isotopes ratio (δ18O and δ2H) were analysed. Exceptionally strong inflow activity from North Sea to the Baltic Sea occurred prior our surveys. The data obtained enabled comprehensive evaluation of tritium and stable isotopes distribution in waters of Baltic Sea and contributing inputs, including riverine and atmospheric freshwater flux and saline water inflow through the Danish Straits. By comparing with the previous studies, we noticed a tendency toward an increase in the δ18O and δ2H values of zero-salinity component over time, which may be due to climate change. In the case of tritium, the influence of artificial sources has also been noticed.

Increased risk of exposure to microcystins in the scum of the filamentous cyanobacterium Aphanizomenon flos-aquae accumulated on the western shoreline of the Curonian Lagoon.

Concentration of toxic cyanobacteria blooms on the downwind shore of high recreational amenity water bodies with largely increases the risk of exposure to cyanotoxins. In this study analysis of phytoplankton structure, cyanotoxin composition and concentration was performed on cyanobacteria scum material, high- and low-density bloom samples in the Curonian Lagoon. We found that the concentration of cyanotoxins in the scum material increased from ∼30 to ∼300-fold compared to bloom samples. In Microcystis aeruginosa dominated samples microcystin-LR was present at the highest concentration, while the dominance of Planktothrix agardhii was associated with the occurrence of dmMC-RR as the major microcystin variant. The toxicological potential of cyanobacterial scums in the Curonian Lagoon is emphasized, and management by removal of these scums is proposed.

Draft Genome Sequence of the Cyanobacterium Aphanizomenon flos-aquae Strain 2012/KM1/D3, Isolated from the Curonian Lagoon (Baltic Sea).

We report here the de novo genome assembly of a cyanobacterium, Aphanizomenon flos-aquae strain 2012/KM1/D3, a harmful bloom-forming species in temperate aquatic ecosystems. The genome is 5.7 Mb with a G+C content of 38.2%, and it is enriched mostly with genes involved in amino acid and carbohydrate metabolism.

Characterization of a lytic cyanophage that infects the bloom-forming cyanobacterium Aphanizomenon flos-aquae.

Vb-AphaS-CL131 is a novel cyanosiphovirus that infects harmful Aphanizomenon flos-aquae. This cyanophage has an isometric head, 97 nm in diameter and a long, flexible non-contractile tail, 361 nm long. With a genome size of ~120 kb, it is the second largest cyanosiphovirus isolated to date. The latent period was estimated to be ~36 h and a single infected cell produces, on average, 218 infectious cyanophages. Cyanophage infection significantly suppresses host biomass production and alters population phenotype.