Nitrogen fixation estimates for the Baltic Sea indicate high rates for the previously overlooked Bothnian Sea.

Dense blooms of diazotrophic filamentous cyanobacteria are formed every summer in the Baltic Sea. We estimated their contribution to nitrogen fixation by combining two decades of cyanobacterial biovolume monitoring data with recently measured genera-specific nitrogen fixation rates. In the Bothnian Sea, estimated nitrogen fixation rates were 80 kt N year-1, which has doubled during recent decades and now exceeds external loading from rivers and atmospheric deposition of 69 kt year-1. The estimated contribution to the Baltic Proper was 399 kt N year-1, which agrees well with previous estimates using other approaches and is greater than the external input of 374 kt N year-1. Our approach can potentially be applied to continuously estimate nitrogen loads via nitrogen fixation. Those estimates are crucial for ecosystem adaptive management since internal nitrogen loading may counteract the positive effects of decreased external nutrient loading.

Cell membrane fatty acid and pigment composition of the psychrotolerant cyanobacterium Nodularia spumigena CHS1 isolated from Hopar glacier, Pakistan.

In the present study, cyanobacterium isolate CHS1 isolated from Hopar glacier, Pakistan, was analyzed for the first time for cell membrane fatty acids and production of pigments. Sequencing of the 16-23S intergenetic region confirmed identification of the isolate CHS1 as Nodularia spumigena. All chlorophyll and carotenoid pigments were quantified using high-performance liquid chromatography and experiments to test tolerance against a range of physico-chemical conditions were conducted. Likewise, the fatty acid profile of the cell membrane CHS1 was analyzed using gas chromatography and mass spectroscopy. The cyanobacterium isolate CHS1 demonstrated tolerance to 8 g/L% NaCl, 35°C and pH 5-9. The characteristic polyunsaturated fatty acid (PUFA) of isolate CHS1, C18:4, was observed in fatty acid methyl esters (FAMEs) extracted from the cell membrane. CHS1 was capable of producing saturated fatty acids (SFA) (e.g., C16:0), monounsaturated fatty acids (MUFA) (e.g., C18:1) and polyunsaturated fatty acids (e.g., C20:5) in the cell membrane. In this study, we hypothesize that one mechanism of cold adaptation displayed by isolate CHS1 is the accumulation of high amounts of PUFA in the cell membrane.

Sedimentation of Nodularia spumigena and distribution of nodularin in the food web during transport of a cyanobacterial bloom from the Baltic Sea to the Kattegat.

Nodularia spumigena is a toxic cyanobacteria that blooms in the Baltic Sea every year. In the brackish water of the Baltic Sea, its toxin, nodularin, mainly affects the biota in the surface water due to the natural buoyancy of this species. However, the fate of the toxin is unknown, once the cyanobacteria bloom enters the more saline waters of the Kattegat. In order to investigate this knowledge gap, a bloom of N. spumigena was followed during its passage, carried by surface currents, from the Baltic Sea into the Kattegat area, through the Öresund strait. N. spumigena cells showed an increased cell concentration through the water column during the passage of the bloom (up to 130 103 cells ml-1), and cells (4.2 103 cells ml-1) could be found down to 20 m depth, below a pycnocline. Sedimentation trap samples from below the pycnocline (10-12 m depth) also showed an increased sedimentation of N. spumigena filaments during the passage of the bloom. The toxin nodularin was detected both in water samples (0.3-6.0 µg l-1), samples of sedimenting material (a toxin accumulation rate of 20 µg m-2 day-1), zooplankton (up to 0.1 ng ind.-1 in copepods), blue mussels (70-230 µg kg-1 DW), pelagic and benthic fish (herring (1.0-3.4 µg kg-1 DW in herring muscle or liver) and flounder (1.3-6.2 µg kg-1 DW in muscle, and 11.7-26.3 µg kg-1 DW in liver). A laboratory experiment showed that N. spumigena filaments developed a decreased buoyancy at increased salinities and that they were even sinking with a rate of up to 1,7 m day-1 at the highest salinity (32 PSU). This has implications for the fate of brackish water cyanobacterial blooms, when these reach more saline waters. It can be speculated that a significant part of the blooms content of nodularin will reach benthic organisms in this situation, compared to blooms decaying in brackish water, where most of the bloom is considered to be decomposed in the surface waters.

Comparative Genomics of the Baltic Sea Toxic Cyanobacteria Nodularia spumigena UHCC 0039 and Its Response to Varying Salinity.

Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spumigena UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.

Niche separation of Baltic Sea cyanobacteria during bloom events by species interactions and autecological preferences.

Cyanobacterial blooms regularly occur in the Baltic Sea during the summer months, with filamentous, heterocystous Nodularia spumigena and Dolichospermum sp. and the coccoid picocyanobacterium Synechococcus spp. as important species. Under calm conditions, N. spumigena accumulate at the surface, whereas Dolichospermum sp. and Synechococcus sp. remain at the subsurface, in the upper water layer. This vertical separation allows co-occurring species to compete for the same resources. The factors that determine the vertical distribution within blooms, however, are mostly unknown. The present study examined the growth rates of these three cyanobacterial species in a two-factorial experiment, with temperature (16 and 24 °C) and radiation (38 and 150 µmol photons m-2 s-1) conditions mimicking those at the water surface and directly below. To determine whether interactions between the three species influenced their growth rates (and therewith also their vertical distribution), paired and multi-species cultures were established. In the single-species cultures, the autecological preferences of the species matched the assumed natural occurrence in bloom events: N. spumigena grew best under high and Dolichospermum sp. and Synechococcus sp. under low light conditions (maximum growth rates at the preferred conditions: µâ€¯= 0.48 ±â€¯0.017, 0.88 ±â€¯0.092, and 0.67 ±â€¯0.012, respectively). High temperatures were tolerated by N. spumigena, but inhibited the growth of Dolichospermum sp. and Synechococcus sp. These results suggested that the factors resulting in the vertical separation of species within a bloom are species-specific: N. spumigena responded predominantly to irradiance and only slightly to temperature, Dolichospermum sp. was intensely affected by temperature and less by irradiance, and Synechococcus sp. responded more strongly to irradiance than to temperature. The interactions in paired and multi-species cultures revealed beneficial and detrimental effects, depending on species composition and abiotic conditions. Under the environmental conditions in which the three species occur, however, similar interactions resulted in no or only slight inhibition. Our observations demonstrate how autecological preferences together with the avoidance of negative interactions determine the vertical distribution of cyanobacteria during bloom events in the Baltic Sea.

Chemical and Genetic Diversity of Nodularia spumigena from the Baltic Sea.

Nodularia spumigena is a toxic, filamentous cyanobacterium occurring in brackish waters worldwide, yet forms extensive recurrent blooms in the Baltic Sea. N. spumigena produces several classes of non-ribosomal peptides (NRPs) that are active against several key metabolic enzymes. Previously, strains from geographically distant regions showed distinct NRP metabolic profiles. In this work, conspecific diversity in N. spumigena was studied using chemical and genetic approaches. NRP profiles were determined in 25 N. spumigena strains isolated in different years and from different locations in the Baltic Sea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genetic diversity was assessed by targeting the phycocyanin intergenic spacer and flanking regions (cpcBA-IGS). Overall, 14 spumigins, 5 aeruginosins, 2 pseudaeruginosins, 2 nodularins, 36 anabaenopeptins, and one new cyanopeptolin-like peptide were identified among the strains. Seven anabaenopeptins were new structures; one cyanopeptolin-like peptide was discovered in N. spumigena for the first time. Based on NRP profiles and cpcBA-IGS sequences, the strains were grouped into two main clusters without apparent influence of year and location, indicating persistent presence of these two subpopulations in the Baltic Sea. This study is a major step in using chemical profiling to explore conspecific diversity with a higher resolution than with a sole genetic approach.

Diversity and Expression of Bacterial Metacaspases in an Aquatic Ecosystem.

Metacaspases are distant homologs of metazoan caspase proteases, implicated in stress response, and programmed cell death (PCD) in bacteria and phytoplankton. While the few previous studies on metacaspases have relied on cultured organisms and sequenced genomes, no studies have focused on metacaspases in a natural setting. We here present data from the first microbial community-wide metacaspase survey; performed by querying metagenomic and metatranscriptomic datasets from the brackish Baltic Sea, a water body characterized by pronounced environmental gradients and periods of massive cyanobacterial blooms. Metacaspase genes were restricted to ~4% of the bacteria, taxonomically affiliated mainly to Bacteroidetes, Alpha- and Betaproteobacteria and Cyanobacteria. The gene abundance was significantly higher in larger or particle-associated bacteria (>0.8 µm), and filamentous Cyanobacteria dominated metacaspase gene expression throughout the bloom season. Distinct seasonal expression patterns were detected for the three metacaspase genes in Nodularia spumigena, one of the main bloom-formers. Clustering of normalized gene expression in combination with analyses of genomic and assembly data suggest functional diversification of these genes, and possible roles of the metacaspase genes related to stress responses, i.e., sulfur metabolism in connection to oxidative stress, and nutrient stress induced cellular differentiation. Co-expression of genes encoding metacaspases and nodularin toxin synthesis enzymes was also observed in Nodularia spumigena. The study shows that metacaspases represent an adaptation of potentially high importance for several key organisms in the Baltic Sea, most prominently Cyanobacteria, and open up for further exploration of their physiological roles in microbes and assessment of their ecological impact in aquatic habitats.

Nodularia spumigena peptides--accumulation and effect on aquatic invertebrates.

Thus far, the negative effects of Nodularia spumigena blooms on aquatic organisms have been mainly attributed to the production of the hepatotoxic nodularin (NOD). In the current work, the accumulation of other N. spumigena metabolites in blue mussels and crustaceans, and their effect on Thamnocephalus platyurus and Artemia franciscana, were examined. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses provided evidence that both blue mussels collected after a cyanobacterial bloom in the Baltic Sea and the crustaceans exposed under laboratory conditions to N. spumigena extract accumulated the cyclic anabaenopeptins (APs). In the crustaceans, the linear peptides, spumigins (SPUs) and aeruginosins (AERs), were additionally detected. Exposure of T. platyurus and A. franciscana to N. spumigena extract confirmed the negative effect of nodularin on the organisms. However, high numbers of dead crustaceans were also recorded in the nodularin-free fraction, which contained protease inhibitors classified to spumigins and aeruginosins. These findings indicate that cyanobacterial toxicity to aquatic organisms is a complex phenomenon and the induced effects can be attributed to diverse metabolites, not only to the known hepatotoxins.

Simple approach for the preparation of (15-15)N2-enriched water for nitrogen fixation assessments: evaluation, application and recommendations.

Recent findings revealed that the commonly used (15)N2 tracer assay for the determination of dinitrogen (N2) fixation can underestimate the activity of aquatic N2-fixing organisms. Therefore, a modification to the method using pre-prepared (15-15)N2-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of (15-15)N2-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add (15-15)N2 gas to the water in amounts exceeding the standard N2 solubility, followed by vigorous agitation (vortex mixing ≥ 5 min). Optionally, water can be degassed at low-pressure (≥950 mbar) for 10 min prior to the (15-15)N2 gas addition to indirectly enhance the (15-15)N2 concentration. This preparation of (15-15)N2-enriched water can be done within 1 h using standard laboratory equipment. The final (15)N-atom% excess was 5% after replacing 2-5% of the incubation volume with (15-15)N2-enriched water. Notably, the addition of (15-15)N2-enriched water can alter levels of trace elements in the incubation water due to the contact of (15-15)N2-enriched water with glass, plastic and rubber ware. In our tests, levels of trace elements (Fe, P, Mn, Mo, Cu, Zn) increased by up to 0.1 nmol L(-1) in the final incubation volume, which may bias rate measurements in regions where N2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with (15-15)N2. The (15-15)N2 was injected as a bubble directly to the incubation water, followed by gentle shaking. Immediately thereafter, the bubble was replaced with water to stop the (15-15)N2 equilibration. This approach achieved a (15)N-atom% excess of 6.6 ± 1.7% when adding 2 mL (15-15)N2 per liter of incubation water. The herein presented methodological tests offer guidelines for the (15)N2 tracer assay and thus, are crucial to circumvent methodological draw-backs for future N2 fixation assessments.

Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria.

Anabaenopeptins, bioactive cyclic hexapeptides, were isolated by preparative reversed-phase high performance liquid chromatography from an extract of Baltic Sea cyanobacterial bloom material composed of Nodularia spumigena (50%), Aphanizomenon flos-aquae (40%) and Dolichospermum spp. (10%). Five new anabaenopeptins and nine previously known anabaenopeptins were isolated, and their putative structures were determined by tandem mass spectrometry. The activity of the peptides against carboxypeptidase A and protein phosphatase 1 as well as chymotrypsin, trypsin and thrombin was tested. All anabaenopeptins inhibited carboxypeptidase A (apart from one anabaenopeptin variant) and protein phosphatase 1 with varying potency, but no inhibition against chymotrypsin, trypsin and thrombin was observed.