Elucidating the picocyanobacteria salinity divide through ecogenomics of new freshwater isolates.

BACKGROUND: Cyanobacteria are the major prokaryotic primary producers occupying a range of aquatic habitats worldwide that differ in levels of salinity, making them a group of interest to study one of the major unresolved conundrums in aquatic microbiology which is what distinguishes a marine microbe from a freshwater one? We address this question using ecogenomics of a group of picocyanobacteria (cluster 5) that have recently evolved to inhabit geographically disparate salinity niches. Our analysis is made possible by the sequencing of 58 new genomes from freshwater representatives of this group that are presented here, representing a 6-fold increase in the available genomic data. RESULTS: Overall, freshwater strains had larger genomes (≈2.9 Mb) and %GC content (≈64%) compared to brackish (2.69 Mb and 64%) and marine (2.5 Mb and 58.5%) isolates. Genomic novelties/differences across the salinity divide highlighted acidic proteomes and specific salt adaptation pathways in marine isolates (e.g., osmolytes/compatible solutes - glycine betaine/ggp/gpg/gmg clusters and glycerolipids glpK/glpA), while freshwater strains possessed distinct ion/potassium channels, permeases (aquaporin Z), fatty acid desaturases, and more neutral/basic proteomes. Sulfur, nitrogen, phosphorus, carbon (photosynthesis), or stress tolerance metabolism while showing distinct genomic footprints between habitats, e.g., different types of transporters, did not obviously translate into major functionality differences between environments. Brackish microbes show a mixture of marine (salt adaptation pathways) and freshwater features, highlighting their transitional nature. CONCLUSIONS: The plethora of freshwater isolates provided here, in terms of trophic status preference and genetic diversity, exemplifies their ability to colonize ecologically diverse waters across the globe. Moreover, a trend towards larger and more flexible/adaptive genomes in freshwater picocyanobacteria may hint at a wider number of ecological niches in this environment compared to the relatively homogeneous marine system.

Primer Design for an Accurate View of Picocyanobacterial Community Structure by Using High-Throughput Sequencing.

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.

Evaluation of the toxicity of leaches from hydrothermal sulfide deposits by means of a delayed fluorescence-based bioassay with the marine cyanobacterium Cyanobium sp. NIES-981.

The commercial use of metals such as copper, lead, and zinc has markedly increased in recent years, resulting in increased interest in deep-sea mining of seafloor hydrothermal sulfide deposits. However, the full extent of the impact of deep-sea mining at hydrothermal field deposits on the environment remains unclear. In addition to impacting the deep sea, the leaching of heavy metals from extracted sulfide mineral may also affect the upper ocean zones as the sulfide rock is retrieved from the seafloor. Here, we used a delayed fluorescence-based bioassay using the marine cyanobacterium Cyanobium sp. NIES-981 to evaluate the toxicity of three sulfide core samples obtained from three drill holes at the Izena Hole, middle Okinawa Trough, East China Sea. Leaches from two of the cores contained high concentrations of zinc and lead, and they markedly inhibited delayed fluorescence in Cyanobium sp. NIES-981 compared with control. By examining the toxicity of artificial mixed-metal solutions with metal compositions similar to those of the leaches, we confirmed that this inhibition was a result of high zinc and lead concentrations into the leaches. In addition, we conclude that this delayed fluorescence-based bioassay is a viable method for use by deep-sea mining operations because it is quicker and requires less laboratory space and equipment than the standard assay.

Microbial F-type lectin domains with affinity for blood group antigens.

F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewisb motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans.

Rapid ecotoxicological bioassay using delayed fluorescence in the marine cyanobacterium Cyanobium sp. (NIES-981).

The use of delayed fluorescence intensity as an endpoint for rapid estimation of the effective concentration (ECx) has been reported as an alternative to standard growth inhibition (at 72 h after exposure) in some algal species including Pseudokirchneriella subcapitata. In marine algae, although an approach of bioassaying using delayed fluorescence measurements has not been performed yet, its development would provide many benefits for marine environmental risk assessment. In this study, we selected marine cyanobacterium Cyanobium sp. (NIES-981) as our test algal species and demonstrated that this species is valid for the standard growth inhibition test based on criteria provide by Organization for Economic Co-operation and Development guidelines. Furthermore, standard inhibition tests and shorter period test using DF were performed in NIES-981 using five chemicals (3,5-DCP, simazine, diflufenican, K2Cr2O7, and CuSO4), and their EC50 and low-toxic-effect values (EC10, EC5, and NOEC) were determined from two dose-response curves. Based on comparisons of the two dose-response curves and the EC50 values, we conclude that DF intensity is useful as an endpoint for rapid estimation of EC50 in NIES-981.

Microcystins and Cyanobacterial Contaminants in the French Small-Scale Productions of Spirulina (Limnospira sp.).

Spirulina is consumed worldwide, in the form of food or dietary supplements, for its nutritional value and health potential. However, these products may contain cyanotoxins, including hepatotoxic microcystins (MCs), produced by cyanobacterial contaminants. The French spirulina market has the particularity of being supplied half-locally by approximately 180 small-scale spirulina production farms. Data about this particular production and possible contaminations with other cyanobacteria and MCs are scarce. Thus, we collected the results of MC analyses and total cyanobacteria counts, carried out between 2013 and 2021, from 95 French spirulina producers who agreed to share their data. These data consisted of MC concentrations determined with an enzyme-linked immunosorbent assay (ELISA) using 623 dry spirulina samples and 105 samples of spirulina cultures. In addition, potentially unsafe samples of dry spirulina were further investigated through mass spectrometry, as duplicate analysis. We confirmed that the situation of the French spirulina production stayed within the safe regulatory level in terms of MC levels. On the other hand, the inventory of cyanobacterial contaminants, based on 539 count results, included 14 taxa. We present their prevalence, interannual evolution and geographical distribution. We also suggested improvements in cultivation practices to limit their propagation.

Shifts in water column microbial composition associated to lakes with different trophic conditions: "Lagunas de Montebello" National Park, Chiapas, México.

Eutrophication is a global problem causing the reduction of water quality and the loss of ecosystem goods and services. The lakes of the "Lagunas de Montebello" National Park (LMNP), Chiapas, Mexico, not only represent unique and beautiful natural scenic sites in southern Mexico but are also a national protected area and RAMSAR site. Unfortunately, some of these lakes started showing eutrophication signs since 2003. Anthropogenic activities (e.g., land-use change from forested to agricultural and urban development) are leading to water quality and trophic state alterations of the lakes of the LMNP. This study shows the results of a coupled limnological characterization and high-throughput sequencing of the V4 hypervariable region of the 16S rRNA gene to analyze the microbial composition of the water column in a set of oligotrophic and eutrophic lakes. Chlorophyll a (Chl-a) was the main environmental parameter correlated with the trophic conditions of the lakes. Although the microbial diversity was similar, the microbial composition changed significantly from oligo to eutrophic lakes. Proteobacteria, Firmicutes, and Cyanobacteria were the main components of oligotrophic lakes, and Cyanobacteria, Proteobacteria, and Bacteroidetes of eutrophic lakes. While Acinetobacter (Proteobacteria) and Cyanobium (a unicellular cyanobacterium) dominated in oligotrophic lakes, the filamentous, bloom-forming, and toxin-producing cyanobacteria Planktothrix was the dominant genus in eutrophic lakes. High-throughput sequencing allowed the detection of changes in the composition of the microbial component in oligotrophic lakes, suggesting a shift towards eutrophication, highlighting the relevance of sensitive monitoring protocols of these ecosystems to implement remediation programs for eutrophicated lakes and conservation strategies for those yet pristine.

Complete Genome Sequences of Chesapeake Bay Synechococcus Strains CBW1002 and CBW1006 Isolated in Winter.

Synechococcus are picocyanobacteria with a cosmopolitan distribution. They are capable of surviving in a wide variety of environmental conditions. Synechococcus have been isolated from the Chesapeake Bay during winter months, and they show an impressive tolerance to cold temperatures. Cold-adapted Synechococcus are unique and diverse, as they have complex phylogenetic lineages closely related to subalpine cluster II, Bornholm Sea cluster, CB7 cluster, and some novel lineages which are independent from summer estuarine strains in subcluster 5.2. CBW1002 and CBW1006 are the first complete genomes to represent Bornholm Sea cluster Synechococcus strains. They have some of the largest genomes among the Synechococcus (3.8 Mb) and share many unique and cryptic homologs which could give insight into their ability to tolerate such cold and dynamic conditions in the Chesapeake Bay estuary.

Metabarcoding Reveals Lacustrine Picocyanobacteria Respond to Environmental Change Through Adaptive Community Structuring.

Picocyanobacteria (Pcy) are important yet understudied components of lake foodwebs. While phylogenetic studies of isolated strains reveal a high diversity of freshwater genotypes, little is known about abiotic drivers associated with Pcy in different lakes. Due to methodological limitations, most previous studies assess potential drivers using total cell abundances as a response, with often conflicting and inconsistent results. In the present study, we explored how picocyanobacterial communities respond to environmental change using a combination of epifluorescence microscopy and community data determined using 16S rRNA gene metabarcoding. Temporal shifts in picocyanobacterial abundance, diversity and community dynamics were assessed in relation to potential environmental drivers in five contrasting lakes over 1year. Cell abundances alone were not consistently related to environmental variables across lakes. However, the addition of metabarcoding data revealed diverse picocyanobacterial communities that differed significantly between lakes, driven by environmental variables related to trophic state. Within each lake, communities were temporally dynamic and certain amplicon sequence variants (ASVs) were strongly associated with specific environmental drivers. Rapid shifts in community structure and composition were often related to environmental changes, indicating that lacustrine Pcy can persist at high abundances through collective community adaptation. These results demonstrate that a combination of microscopy and metabarcoding enables an in-depth characterisation of picocyanobacterial communities and reveals strain-specific drivers. We recommend that future studies cease referring to picocyanobacterial as one functional group and take strain specific variability into consideration.

Spatial abundance and distribution of picocyanobacterial communities in two contrasting lakes revealed using environmental DNA metabarcoding.

Freshwater picocyanobacteria (Pcy) are important yet understudied components of lake ecosystems. Most previous studies have relied on cell abundances to assess Pcy dynamics in largely oligotrophic lakes, while little is known about spatial diversity and dynamics across different lake types. In the present study we assessed the horizontal-spatial abundance and community structure of Pcy in two contrasting (oligotrophic and hypertrophic) New Zealand lakes using epifluorescence microscopy and 16S rRNA metabarcoding. Pcy abundance and community composition differed significantly both between and within the oligotrophic and hypertrophic lakes. While spatial variability was observed in both study lakes, these differences were particularly pronounced in the oligotrophic, morphometrically complex Lake Wanaka where cell abundances were typically higher in bays than open-water sites and community structure differed significantly between sites. Community structuring appeared to be driven by localised environmental conditions, with different factors influencing each lake. These results suggest that single spot-samples are insufficient to gain an understanding of Pcy dynamics and consequently, phytoplankton dynamics in lakes.

Bioactive Potential of Two Marine Picocyanobacteria Belonging to Cyanobium and Synechococcus Genera.

Coccoid cyanobacteria produce a great variety of secondary metabolites, which may have useful properties, such as antibacterial, antiviral, anticoagulant or anticancer activities. These cyanobacterial metabolites have high ecological significance, and they could be considered responsible for the widespread occurrence of these microorganisms. Considering the great benefit derived from the identification of competent cyanobacteria for the extraction of bioactive compounds, two strains of picocyanobacteria (coccoid cyanobacteria < 3 µm) (Cyanobium sp. ITAC108 and Synechococcus sp. ITAC107) isolated from the Mediterranean sponge Petrosia ficiformis were analyzed. The biological effects of organic and aqueous extracts from these picocyanobacteria toward the nauplii of Artemia salina, sea urchin embryos and human cancer lines (HeLa cells) were evaluated. Methanolic and aqueous extracts from the two strains strongly inhibited larval development; on the contrary, in ethyl acetate and hexane extracts, the percentage of anomalous embryos was low. Moreover, all the extracts of the two strains inhibited HeLa cell proliferation, but methanol extracts exerted the highest activity. Gas chromatography-mass spectrometry analysis evidenced for the first time the presence of ß-N-methylamino-l-alanine and microcystin in these picocyanobacteria. The strong cytotoxic activity observed for aqueous and methanolic extracts of these two cyanobacteria laid the foundation for the production of bioactive compounds of pharmacological interest.

Species-Level Spatio-Temporal Dynamics of Cyanobacteria in a Hard-Water Temperate Lake in the Southern Baltics.

Cyanobacteria are important primary producers in temperate freshwater ecosystems. However, studies on the seasonal and spatial distribution of cyanobacteria in deep lakes based on high-throughput DNA sequencing are still rare. In this study, we combined monthly water sampling and monitoring in 2019, amplicon sequence variants analysis (ASVs; a proxy for different species) and quantitative PCR targeting overall cyanobacteria abundance to describe the seasonal and spatial dynamics of cyanobacteria in the deep hard-water oligo-mesotrophic Lake Tiefer See, NE Germany. We observed significant seasonal variation in the cyanobacterial community composition (p < 0.05) in the epi- and metalimnion layers, but not in the hypolimnion. In winter-when the water column is mixed-picocyanobacteria (Synechococcus and Cyanobium) were dominant. With the onset of stratification in late spring, we observed potential niche specialization and coexistence among the cyanobacteria taxa driven mainly by light and nutrient dynamics. Specifically, ASVs assigned to picocyanobacteria and the genus Planktothrix were the main contributors to the formation of deep chlorophyll maxima along a light gradient. While Synechococcus and different Cyanobium ASVs were abundant in the epilimnion up to the base of the euphotic zone from spring to fall, Planktothrix mainly occurred in the metalimnetic layer below the euphotic zone where also overall cyanobacteria abundance was highest in summer. Our data revealed two potentially psychrotolerant (cold-adapted) Cyanobium species that appear to cope well under conditions of lower hypolimnetic water temperature and light as well as increasing sediment-released phosphate in the deeper waters in summer. The potential cold-adapted Cyanobium species were also dominant throughout the water column in fall and winter. Furthermore, Snowella and Microcystis-related ASVs were abundant in the water column during the onset of fall turnover. Altogether, these findings suggest previously unascertained and considerable spatiotemporal changes in the community of cyanobacteria on the species level especially within the genus Cyanobium in deep hard-water temperate lakes.

Microbial community successions and their dynamic functions during harmful cyanobacterial blooms in a freshwater lake.

The current study reports the community succession of different toxin and non-toxin producing cyanobacteria at different stages of cyanobacterial harmful algal blooms (CyanoHABs) and their connectivity with nitrogen and phosphorus cycles in a freshwater lake using an ecogenomics framework. Comprehensive high throughput DNA sequencing, water quality parameter measurements, and functional gene expressions over temporal and spatial scales were employed. Among the cyanobacterial community, the lake was initially dominated by Cyanobium during the months of May, June, and early July, and later primarily by Aphanizomenon and Dolichospermum depicting functional redundancy. Finally, Planktothrix appeared in late August and then the dominance switched to Planktothrix in September. Microcystis aeruginosa and Microcystis panniformis; two species responsible for cyanotoxin production, were also present in August and September, but in significantly smaller relative abundance. MC-LR (0.06-1.32 µg/L) and MC-RR (0.01-0.26 µg/L) were two major types of cyanotoxins detected. The presence of MC-LR and MC-RR were significantly correlated with the Microcystis-related genes (16SMic/mcyA/mcyG) and their expressions (r = 0.33 to 0.8, p < 0.05). The metabolic analyses further linked the presence of different cyanobacterial groups with distinct functions. The nitrogen metabolisms detected a relatively higher abundance of nitrite/nitrate reductase in early summer, indicating significant denitrification activity and the activation of N-fixation in the blooms dominated by Aphanizomenon/Dolichospermum (community richness) during nutrient-limited conditions. The phosphorus and carbohydrate metabolisms detected a trend to initiate a nutrient starvation alert and store nutrients from early summer, while utilizing the stored polyphosphate and carbohydrate (PPX and F6PPK) during the extreme ortho-P scarcity period, mostly in August or September. Specifically, the abundance of Aphanizomenon and Dolichospermum was positively correlated with the nitrogen-fixing nif gene and (p < 0.001) and the PPX enzyme for the stored polyphosphate utilization (r = 0.77, p < 0.001). Interestingly, the lake experienced a longer N-fixing period (2-3 months) before non-fixing cyanobacteria (Planktothrix) dominated the entire lake in late summer. The Provo Bay site, which is known to be nutrient-rich historically, had early episodes of filamentous cyanobacteria blooms compared to the rest of the lake.

A Large-Scale Survey of the Bacterial Communities in Lakes of Western Mongolia with Varying Salinity Regimes.

In recent years, climate change coupled with anthropogenic activities has led to monumental changes in saline lakes which are rapidly drying up across the globe and particularly in Central Asia. The landlocked country of Mongolia is rich in lakes which have remained primarily undisturbed by human impact, and many of these lakes have varying salinity regimes and are located across various geographical landscapes. In this study, we sampled 18 lakes with varying salinity regimes (hyperhaline, mesohaline, oligohaline, and polyhaline) covering 7000 km of western Mongolia and its various geographical landscapes (Gobi Desert, forests, and steppe). We identified that the bacterial communities that dominate these lakes are significantly influenced by salinity (p < 0.001) and geographical landscape (p < 0.001). Further, only five zOTUs were shared in all the lakes across the salinity regimes, providing evidence that both local and regional factors govern the community assembly and composition. Furthermore, the bacterial communities of hyperhaline lakes were significantly positively correlated with salinity (ANOVA, p < 0.001) and arsenic concentrations (ANOVA, p < 0.001), whereas bacterial communities of mesohaline and polyhaline lakes situated in forest and steppe landscapes were positively correlated with temperature (ANOVA, p < 0.001) and altitude (ANOVA, p < 0.001), respectively. Functional predictions based on the 16S rRNA gene indicated enrichment of KEGG Ontology terms related to transporters for osmoprotection and -regulation. Overall, our study provides a comprehensive view of the bacterial diversity and community composition present in these lakes, which might be lost in the future.

Insights Into the Evolution of Picocyanobacteria and Phycoerythrin Genes (mpeBA and cpeBA).

Marine picocyanobacteria, Prochlorococcus and Synechococcus, substantially contribute to marine primary production and have been the subject of extensive ecological and genomic studies. Little is known about their close relatives from freshwater and non-marine environments. Phylogenomic analyses (using 136 proteins) provide strong support for the monophyly of a clade of non-marine picocyanobacteria consisting of Cyanobium, Synechococcus and marine Sub-cluster 5.2; this clade itself is sister to marine Synechococcus and Prochlorococcus. The most basal lineage within the Syn/Pro clade, Sub-Cluster 5.3, includes marine and freshwater strains. Relaxed molecular clock (SSU, LSU) analyses show that while ancestors of the Syn/Pro clade date as far back as the end of the Pre-Cambrian, modern crown groups evolved during the Carboniferous and Triassic. Comparative genomic analyses reveal novel gene cluster arrangements involved in phycobilisome (PBS) metabolism in freshwater strains. Whilst PBS genes in marine Synechococcus are mostly found in one type of phycoerythrin (PE) rich gene cluster (Type III), strains from non-marine habitats, so far, appear to be more diverse both in terms of pigment content and gene arrangement, likely reflecting a wider range of habitats. Our phylogenetic analyses show that the PE genes (mpeBA) evolved via a duplication of the cpeBA genes in an ancestor of the marine and non-marine picocyanobacteria and of the symbiotic strains Synechococcus spongiarum. A 'primitive' Type III-like ancestor containing cpeBA and mpeBA had thus evolved prior to the divergence of the Syn/Pro clade and S. spongiarum. During the diversification of Synechococcus lineages, losses of mpeBA genes may explain the emergence of pigment cluster Types I, II, IIB, and III in both marine and non-marine habitats, with few lateral gene transfer events in specific taxa.

Identification of Cyanobacteria in a Eutrophic Coastal Lagoon on the Southern Baltic Coast.

Cyanobacteria are found worldwide in various habitats. Members of the picocyanobacteria genera Synechococcus and Prochlorococcus dominate in oligotrophic ocean waters. Other picocyanobacteria dominate in eutrophic fresh or brackish waters. Usually, these are morphologically determined as species of the order Chroococcales/clade B2. The phytoplankton of a shallow, eutrophic brackish lagoon was investigated. Phytoplankton was dominated by Aphanothece-like morphospecies year-round for more than 20 years, along a trophy and salinity gradient. A biphasic approach using a culture-independent and a culture-dependent analysis was applied to identify the dominant species genetically. The 16S rRNA gene phylogeny of clone sequences and isolates indicated the dominance of Cyanobium species (order Synechococcales sensu Komárek/clade C1 sensu Shih). This difference between morphologically and genetically based species identifications has consequences for applying the Reynolds functional-groups system, and for validity long-term monitoring data. The literature shows the same pattern as our results: morphologically, Aphanothece-like species are abundant in eutrophic shallow lagoons, and genetically, Cyanobium is found in similar habitats. This discrepancy is found worldwide in the literature on fresh- and brackish-water habitats. Thus, most Aphanothece-like morphospecies may be, genetically, members of Cyanobium.