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Summary 449 I. INTRODUCTION 450 II. THE PARTNERS 451 1. Cyanobionts and their role 451 2. Hosts and their role 453 3. Location of cyanobionts in their hosts 455 III. INITIATION AND DEVELOPMENT OF SYMBIOSES 458 1. Initiation of symbioses 458 2. Geosiphon pyriforme 458 3. Cyanolichens 459 4. Liverworts and hornworts 460 5. Azolla 460 6. Cycads 461 7. Gunnera 461 IV. THE SYMBIOSES 462 1. Geographical distribution and ecological significance 462 2. Benefits to the partners 462 (a) Benefits to the cyanobionts 462 (b) Benefits to the hosts 463 3. Duration and stability 463 4. Mode of transmission and perpetuation 463 5. Recognition between the partners 464 6. Specificity and diversity 464 7. Symbiosis-related genes 465 8. Modifications of the cyanobiont 466 (a) Growth and morphology 466 (b) Photosynthesis and carbon metabolism 467 (c) Glutamine synthetase 467 (d) Heterocysts 469 (e) N2fixation 470 9. Nutrient exchange 471 (a) Carbon 471 (b) Nitrogen 472 V. EVOLUTIONARY ASPECTS 472 VI. ARTIFICIAL SYMBIOSES 474 VII. FUTURE OUTLOOK AND PERSPECTIVES 475 1. Cryptic symbioses 476 2. Developmental profile of symbiotic tissues 476 3. Sensing and signalling 476 4. Genetic aspects 476 5. Physiological and biochemical aspects of nutrient exchange 477 6. Microaerobiosis 477 7. Potential applications 477 Acknowledgements 477 References 477 Cyanobacteria are an ancient, morphologically diverse group of prokaryotes with an oxygenic photosynthesis. Many cyanobacteria also possess the ability to fix N2. Although well suited to an independent existence in nature, some cyanobacteria occur in symbiosis with a wide range of hosts (protists, animals and plants). Among plants, such symbioses have independently evolved in phylogenetically diverse genera belonging to the algae, fungi, bryophytes, pteridophytes, gymnosperms and angiosperms. These are N2-fixing symbioses involving heterocystous cyanobacteria, particularly Nostoc, as cyanobionts (cyanobacterial partners). A given host species associates with only a particular cyanobiont genus but such specificity does not extend to the strain level. The cyanobiont is located under a microaerobic environment in a variety of host organs and tissues (bladder, thalli and cephalodia in fungi; cavities in gametophytes of hornworts and liverworts or fronds of the Azolla sporophyte; coralloid roots in cycads; stem glands in Gunnera). Except for fungi, the hosts form these structures ahead of the cyanobiont infection. The symbiosis lasts for one generation except in Azolla and diatoms, in which it is perpetuated from generation to generation. Within each generation, multiple fresh infections occur as new symbiotic tissues and organs develop. The symbioses are stable over a wide range of environmental conditions, and sensing–signalling between partners ensures their synchronized growth and development. The cyanobiont population is kept constant in relation to the host biomass through controlled initiation and infection, nutrient supply and cell division. In most cases, the partners have remained facultative, with the cyanobiont residing extracellularly in the host. However, in the water-fern Azolla and the freshwater diatom Rhopalodia the association is obligate. The cyanobionts occur intracellularly in diatoms, the fungus Geosiphon and the angiosperm Gunner a. Close cell–cell contact and the development of special structures ensure efficient nutrient exchange between the partners. The mobile nutrients are normal products of the donor cells, although their production is increased in symbiosis. Establishment of cyanobacterial–plant symbioses differs from chloroplast evolution. In these symbioses, the cyanobiont undergoes structural–functional changes suited to its role as provider of fixed N rather than fixed C, and the level of intimacy is far less than that of an organelle. This review provides an updated account of cyanobacterial–plant symbioses, particularly concerning developments during the past 10 yr. Various aspects of these symbioses such as initiation and development, symbiont diversity, recognition and signalling, structural–functional modifications, integration, and nutrient exchange are reviewed and discussed, as are evolutionary aspects and the potential uses of cyanobacterial–plant symbioses. Finally we outline areas that require special attention for future research. Not only will these provide information of academic interest but they will also help to improve the use of Azolla as green manure, to enable us to establish artificial N2-fixing associations with cereals such as rice, and to allow the manipulation of free-living cyanobacteria for photobiological ammonia or hydrogen production or for use as biofertilizers.

Lake Big Momela, one of the East African soda lakes in Northern Tanzania characterised by highly saline-alkaline conditions, making them inhospitable to a range of organisms, although supporting massive growths of some adapted planktonic microorganisms that serve as food for birds, such as Lesser Flamingo. The temporal dynamics of plankton, with an emphasis on cyanobacteria, were examined in 2007 using morphological traits and ribosomal genetic markers (16S and 18S rRNA). Cyanobacterial genes encoding for hepatotoxins (mcyE and ndaF) were also screened. Rotifers and copepods dominated the zooplankton, whereas cyanobacteria, such as Anabaenopsis elenkinii and Arthrospira fusiformis dominated the phytoplankton community, and these being related to representatives in other East African soda lakes. The cyanobacteria community also showed distinct seasonal patterns influenced by environmental parameters, mainly salinity, pH and nitrate. Significant positive correlations were found between phytoplankton abundance and nitrate concentrations (r = 0.617, p = 0.033). No signals of the hepatotoxin synthetase genes mcyE and ndaF were retrieved from cyanobacteria during the whole year. In general, our data illustrate the presence of rich planktonic communities, including some unique and potentially endemic cyanobacteria.Keywords: cyanotoxin, limnology, plankton diversity, soda lakes

Cyanobacteria are widely distributed primary producers with significant implications for the global biogeochemical cycles of carbon and nitrogen. Diazotrophic cyanobacteria of subsection V (Order Stigonematales) are particularly ubiquitous in photoautotrophic microbial mats of hot springs. The Stigonematal cyanobacterium strain CHP1 isolated from the Porcelana hot spring (Chile) was one of the major contributors of the new nitrogen through nitrogen fixation. Further morphological and genetic characterization verified that the strain CHP1 belongs to Stigonematales, and it formed a separate clade together with other thermophiles of the genera Fischerella and Mastigocladus. Strain CHP1 fixed maximum N2 in the light, independent of the temperature range. At 50°C nifH gene transcripts showed high expression during the light period, whereas the nifH gene expression at 45°C was arrhythmic. The strain displayed a high affinity for nitrate and a low tolerance for high ammonium concentrations, whereas the narB and glnA genes showed higher expression in light and at the beginning of the dark phase. It is proposed that Mastigocladus sp. strain CHP1 would represent a good model for the study of subsection V thermophilic cyanobacteria, and for understanding the adaptations of these photoautotrophic organisms inhabiting microbial mats in hot springs globally.

Cyanobacteria (blue-green algae) are photosynthetic prokaryotes with an oxygenic photosynthesis. Reproduction in cyanobacteria is generally by fission, budding, trichome breakage, hormogonia formation, or akinete germination. Cyanobacteria form symbiotic associations with plants, animals, nonphotosynthetic protists belonging to the group Glaucophyta, and bacteria. Among plants, cyanobacteria form symbiotic associations with algae (diatoms), fungi (lichens), bryophytes (liverworts, hornworts, and mosses), pteridophytes, gymnosperms, and angiosperme. In all other plant symbioses, the cyanobiont is a heterocystous filamentous cyanobacterium belonging to the genus Nostoc or Anabaena. Cyanobacterial-algal symbioses, together with the cyanobacterial symbioses with animals, glaucophytes, and bacteria. Two groups of animals are reported to have cyanobacterial symbionts: echiruoid worms and marine sponges. The bacterial symbionts are often concentrated around the heterocysts or the heterocysts-vegetative cell junctions. The incidence of symbiosis is very common in sponges occurring in the tropical marine environment and it has a fairly wide geographical distribution.

Marine and brackish surface waters are highly dynamic habitats that undergo repeated seasonal variations in microbial community composition and function throughout time. While succession of the various microbial groups has been well investigated, little is known about the underlying gene-expression of the microbial community. We investigated microbial interactions via metatranscriptomics over a spring to fall seasonal cycle in the brackish Baltic Sea surface waters, a temperate brackish water ecosystem periodically promoting massive cyanobacterial blooms, which have implications for e.g. primary production, nutrient cycling, and expansion of hypoxic zones. Network analysis of the gene expression of all microbes from 0.22 - 200 µm in size and of the major taxonomic groups dissected the seasonal cycle into four components that comprised genes peaking during different periods of the bloom. Photoautotrophic nitrogen-fixing Cyanobacteria displayed the highest connectivity among the microbes, in contrast to chemoautotrophic ammonia-oxidizing Thaumarchaeota, while heterotrophs dominated connectivity among pre- and post-bloom peaking genes. The network was also composed of distinct functional connectivities, with an early season balance between carbon metabolism and ATP synthesis shifting to a dominance of ATP synthesis during the bloom, while carbon degradation, specifically through the glyoxylate shunt, characterized the post-bloom period, driven by Alphaproteobacteria as well as by Gammaproteobacteria of the SAR86 and SAR92 clusters. Our study stresses the exceptionally strong biotic driving force executed by cyanobacterial blooms on associated microbial communities in the Baltic Sea and highlights the impact cyanobacterial blooms have on functional microbial community composition.

Inferred virus-host relationships, community structures of ubiquitous ecologically relevant groups, and identification of transcriptionally active populations have been achieved with our Baltic Sea study. Further, these data, highlighting the transcriptional activity of viruses, represent one of the more powerful uses of omics concerning ecosystem health. The use of omics-related data to assess ecosystem health holds great promise for rapid and relatively inexpensive determination of perturbations and risk, explicitly with regard to viral assemblages, as no single marker gene is suitable for widespread taxonomic coverage., Metagenomic and metatranscriptomic data were generated from size-fractionated samples from 11 sites within the Baltic Sea and adjacent marine waters of Kattegat and freshwater Lake Torneträsk in order to investigate the diversity, distribution, and transcriptional activity of virioplankton. Such a transect, spanning a salinity gradient from freshwater to the open sea, facilitated a broad genome-enabled investigation of natural as well as impacted aspects of Baltic Sea viral communities. Taxonomic signatures representative of phages within the widely distributed order Caudovirales were identified with enrichments in lesser-known families such as Podoviridae and Siphoviridae. The distribution of phage reported to infect diverse and ubiquitous heterotrophic bacteria (SAR11 clades) and cyanobacteria (Synechococcus sp.) displayed population-level shifts in diversity. Samples from higher-salinity conditions (>14 practical salinity units [PSU]) had increased abundances of viruses for picoeukaryotes, i.e., Ostreococcus. These data, combined with host diversity estimates, suggest viral modulation of diversity on the whole-community scale, as well as in specific prokaryotic and eukaryotic lineages. RNA libraries revealed single-stranded DNA (ssDNA) and RNA viral populations throughout the Baltic Sea, with ssDNA phage highly represented in Lake Torneträsk. Further, our data suggest relatively high transcriptional activity of fish viruses within diverse families known to have broad host ranges, such as Nodoviridae (RNA), Iridoviridae (DNA), and predicted zoonotic viruses that can cause ecological and economic damage as well as impact human health. IMPORTANCE Inferred virus-host relationships, community structures of ubiquitous ecologically relevant groups, and identification of transcriptionally active populations have been achieved with our Baltic Sea study. Further, these data, highlighting the transcriptional activity of viruses, represent one of the more powerful uses of omics concerning ecosystem health. The use of omics-related data to assess ecosystem health holds great promise for rapid and relatively inexpensive determination of perturbations and risk, explicitly with regard to viral assemblages, as no single marker gene is suitable for widespread taxonomic coverage.

Cyanobacteria produce a range of secondary metabolites, one being the neurotoxic non-protein amino acid β-N-methylamino-L-alanine (BMAA), proposed to be a causative agent of human neurodegeneration. As for most cyanotoxins, the function of BMAA in cyanobacteria is unknown. Here, we examined the effects of BMAA on the physiology of the filamentous nitrogen-fixing cyanobacterium Nostoc sp. PCC 7120. Our data show that exogenously applied BMAA rapidly inhibits nitrogenase activity (acetylene reduction assay), even at micromolar concentrations, and that the inhibition was considerably more severe than that induced by combined nitrogen sources and most other amino acids. BMAA also caused growth arrest and massive cellular glycogen accumulation, as observed by electron microscopy. With nitrogen fixation being a process highly sensitive to oxygen species we propose that the BMAA effects found here may be related to the production of reactive oxygen species, as reported for other organisms.

The last several decades have witnessed dramatic advances in unfolding the diversity and commonality of oceanic diazotrophs and their N2 -fixing potential. More recently, substantial progress in diazotrophic cell biology has provided a wealth of information on processes and mechanisms involved. The substantial contribution by the diazotrophic cyanobacterial genus Trichodesmium to the nitrogen influx of the global marine ecosystem is by now undisputable and of paramount ecological importance, while the underlying cellular and molecular regulatory physiology has only recently started to unfold. Here, we explore and summarize current knowledge, related to the optimization of its diazotrophic capacity, from genomics to ecophysiological processes, via, for example, cellular differentiation (diazocytes) and temporal regulations, and suggest cellular research avenues that now ought to be explored.

Seagrasses are photoautotrophic, ecologically important components of many globally widespread coastal ecosystems, in which combined nitrogen may limit their production. We examined the biodiversity and diazotrophic capacity of microbial epiphytes associated with the phyllosphere of the seagrass Cymodocea rotundata of the Western Indian Ocean. Light microscopy, 16S rRNA and nifH gene analysis revealed the dominance of cyanobacteria in the epiphytic microbial community. Most phylotypes were related to free-living uncultured benthic cyanobacteria, while some to cyanobacterial endosymbionts of marine diatoms. Novel and potentially diazotrophic species, some of known pantropical distribution, were also discovered. Significant diel nitrogenase activities (acetylene reduction assay) were recorded (up to 358 ± 232 nmol C2H4 g(-1) of seagrass FW h(-1)). The nifH gene expression patterns showed that heterocystous phylotypes may be the dominant diazotrophs during the day and non-heterocystous at night. These data show that C. rotundata is colonized by diverse diazotrophic cyanobacteria species and suggest that these may be beneficial partners of seagrasses in nitrogen-depleted waters.

Cyanobacteria are important phytoplankton in the Baltic Sea, an estuarine-like environment with pronounced north to south gradients in salinity and nutrient concentrations. Here, we present a metagenomic and -transcriptomic survey, with subsequent analyses targeting the genetic identity, phylogenetic diversity, and spatial distribution of Baltic Sea cyanobacteria. The cyanobacterial community constituted close to 12% of the microbial population sampled during a pre-bloom period (June-July 2009). The community was dominated by unicellular picocyanobacteria, specifically a few highly abundant taxa (Synechococcus and Cyanobium) with a long tail of low abundance representatives, and local peaks of bloom-forming heterocystous taxa. Cyanobacteria in the Baltic Sea differed genetically from those in adjacent limnic and marine waters as well as from cultivated and sequenced picocyanobacterial strains. Diversity peaked at brackish salinities 3.5-16 psu, with low N:P ratios. A shift in community composition from brackish to marine strains was accompanied by a change in the repertoire and expression of genes involved in salt acclimation. Overall, the pre-bloom cyanobacterial population was more genetically diverse, widespread and abundant than previously documented, with unicellular picocyanobacteria being the most abundant clade along the entire Baltic Sea salinity gradient.

Rhodopsins are light-driven ion-pumping membrane proteins found in many organisms and are proposed to be of global importance for oceanic microbial energy generation. Several studies have focused on marine environments, with less exploration of rhodopsins in brackish waters. We investigated microbial rhodopsins in the Baltic Sea using size-fractionated metagenomic and metatranscriptomic datasets collected along a salinity gradient spanning from ∼0 to 35 PSU. The normalised genomic abundance of rhodopsins in Bacteria, as well as rhodopsin gene expression, was highest in the smallest size fraction (0.1-0.8 μm), relative to the medium (0.8-3.0 μm) and large (3.0 μm) size fractions. The abundance of rhodopsins in the two smaller size fractions displayed a positive correlation with salinity. Proteobacteria and Bacteroidetes rhodopsins were the most abundant while Actinobacteria rhodopsins, or actinorhodopsins, were common at lower salinities. Phylogenetic analysis indicated that rhodopsins have adapted independently to the marine-brackish transition on multiple occasions, giving rise to green light-adapted variants from ancestral blue light-adapted ones. A notable diversity of viral-like rhodopsins was also detected in the dataset and potentially linked with eukaryotic phytoplankton blooms. Finally, a new clade of likely proton-pumping rhodopsin with non-canonical amino acids in the spectral tuning and proton accepting site was identified.

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.

International audience; Cyanobacteria from Subsection V (Stigonematales) are important components of microbial mats in non-acidic terrestrial hot springs. Despite their diazotrophic nature (N2 fixers), their impact on the nitrogen cycle in such extreme ecosystems remains unknown. Here, we surveyed the identity and activity of diazotrophic cyanobacteria in the neutral hot spring of Porcelana (Northern Patagonia, Chile) during 2009 and 2011–2013. We used 16S rRNA and the nifH gene to analyze the distribution and diversity of diazotrophic cyanobacteria. Our results demonstrate the dominance of the heterocystous genus Mastigocladus (Stigonematales) along the entire temperature gradient of the hot spring (69–38 °C). In situ nitrogenase activity (acetylene reduction), nitrogen fixation rates (cellular uptake of 15N2) and nifH transcription levels in the microbial mats showed that nitrogen fixation and nifH mRNA expression were light-dependent. Nitrogen fixation activities were detected at temperatures ranging from 58 °C to 46 °C, with maximum daily rates of 600 nmol C2H4 cm−2 per day and 94.1 nmol N cm−2 per day. These activity patterns strongly suggest a heterocystous cyanobacterial origin and reveal a correlation between nitrogenase activity and nifH gene expression during diurnal cycles in thermal microbial mats. N and C fixation in the mats contributed ~3 g N m−2 per year and 27 g C m−2 per year, suggesting that these vital demands are fully met by the diazotrophic and photoautotrophic capacities of the cyanobacteria in the Porcelana hot spring.

Although cyanobacterial diazotrophs are common in Arctic terrestrial and freshwater habitats, they have been assumed to be absent from Arctic marine habitats. We report here a high diversity of cyanobacterial nifH genes in Fram Strait and the Greenland Sea. The nifH gene encodes the iron protein of the nitrogenase enzyme complex, which is essential for biological N2 fixation. Using primers specific for nifH genes we uncovered communities of autotrophic and heterotrophic bacteria in sea ice brine and seawater between latitudes 65 and 81°N. Cyanobacteria (Oscillatoriales and Chroococcales) with known marine planktonic and benthic distributions were distinguished, alongside a mix of metabolically versatile eubacteria (nifH Clusters I and III). Using primers selective for cyanobacterial nifH genes we identified filamentous non-heterocystous Trichodesmium-like and LPP (Leptolyngbya, Phormidium and Plectonema)-like Oscillatoriales, as well as Cyanothece-like Chroococcales in a brine sample from 81°N. The occurrence of Trichodesmium-like cyanobacteria was further confirmed by sequences of the hetR gene of Trichodesmium. Microscopic examinations confirmed the presence of viable filamentous and unicellular cyanobacteria. Our results reveal the potential for microbial N2 fixation in the Arctic seas. However, it is still left to determine if these genes are also metabolically active before any biogeochemical importance of diazotrophy in the polar oceans can be assessed.

The establishment of non-diazotrophic cultures of the filamentous marine cyanobacterium Trichodesmium erythraeum IMS101 enabled the first detailed investigation of the process leading to the development of its unique nitrogen-fixing cell type, the diazocyte. Trichome heterogeneity was apparent already within 3-8 h, while the differentiation of mature diazocytes, containing the nitrogenase enzyme, required 27 h after the removal of combined nitrogen. The distribution of 'pro-diazocytes' within the trichomes correlates with the localization of mature diazocytes, which suggests that pattern regulation is an early event during diazocyte development. The development was initially identified as changes in the subcellular ultrastructure, most notably the degradation of glycogen granules and gas vacuoles. These changes were preceded by the induced expression of the global nitrogen regulator ntcA at an early stage of combined nitrogen deprivation, followed by elevated expression of genes related to nitrogen metabolism and their corresponding proteins. The strongest induction (10-fold) was related to the transcription of the respiratory gene coxB2, apparent already at an early stage, which suggests an important role for respiration and the subsequent energy generation in the subcellular changes found, and in the creation of the reducing environment required for nitrogen fixation in diazocytes.

The annually reoccurring blooms that characterize the surface waters of the Baltic Sea are dominated by filamentous, heterocystous cyanobacteria such as Nodularia spumigena. In a previous study, we have demonstrated that N. spumigena strain AV1 differentiates heterocysts in the absence of detectable nitrogen fixation activity, an unusual physiological trait that is clearly distinct from other well-studied cyanobacteria. To further analyze the uncoupling between these two processes, we analyzed the gene expression and modification of the nitrogenase enzyme (the enzyme responsible for nitrogen fixation) in N. spumigena AV1, as well as in several other N. spumigena strains. Here, we demonstrate the occurrence of two nifH gene copies in N. spumigena strain AV1, only one of which is located in a complete nifHDK cluster and several NifH protein forms. Furthermore, we demonstrate the occurrence of a DNA rearrangement mechanism acting within the nifH gene copy located in the nifHDK cluster and present only in the strains exhibiting the previously reported uncoupling between heterocyst differentiation and nitrogen fixation processes. These data stress the existence of a distinct and complex regulatory circuit related to nitrogen fixation in this ecologically significant bloom-forming cyanobacterium.

Heterocyst-forming cyanobacteria are important players at both evolutionary and ecological scales, but to date it has been difficult to establish their phylogenetic affiliations. We present data from a phylogenetic and molecular clock analysis of heterocystous cyanobacteria within the family Rivulariaceae, including the genera Calothrix, Rivularia, Gloeotrichia and Tolypothrix. The strains were isolated from distant geographic regions including fresh and brackish water bodies, microbial mats from beach rock, microbialites, pebble beaches, plus PCC strains 7103 and 7504. Phylogenetic inferences (distance, likelihood and Bayesian) suggested the monophyly of genera Calothrix and Rivularia. Molecular clock estimates indicate that Calothrix and Rivularia originated ∼1500 million years ago (MYA) ago and species date back to 400-300 MYA while Tolypothrix and Gloeotrichia are younger genera (600-400 MYA).

Tropical seagrass ecosystems are highly productive and important for sustaining marine life and associated coastal societies. In this study, the diversity and role of nitrogen-fixing cyanobacteria associated with five common seagrass genera in coastal regions of the western Indian Ocean (WIO; Tanzania) were examined, as well as the impact of anthropogenic activities. Cyanobacteria were characterized morphologically and genetically (16S rRNA and nifH gene phylogeny), as were diel variations in nifH gene expression, NifH protein levels and nitrogenase activity. The results revealed that WIO seagrass beds supported rich cyanobacterial diversity and that these represented approx. 83% of total clones obtained (DNA and RNA nifH clone libraries). Non-heterocystous genera, such as Oscillatoria, Lyngbya, Leptolyngbya, Phormidium and Microcoleus dominated, while heterocystous morphotypes such as Calothrix were less frequent and unicellular morphotypes (e.g. Gloeocapsa, Chroococcus and Chroococcidiopsis) were few. Additionally, the phylogenetic analysis revealed several novel uncharacterized cyanobacterial clades. Cyanobacterial composition and nitrogenase activity varied over seasons and between the seagrass species. Day time nitrogenase activity originated primarily from heterocystous phylotypes, while non-heterocystous filamentous phylotypes fixed nitrogen at night. The highest activity in the diel cycle was 358 ± 232 nmol C2H4 g-1 h-1at 09.00 associated with epiphytes of the seagrass Cymodocea. Nitrogenase activity was consistently lower in anthropogenically disturbed (eutrophication) seagrass sites. Such data suggest that diazotrophic cyanobacteria may be a significant source of ‘new’ nitrogen in the often oligotrophic coastal regions of tropical oceans. It is also proposed that the rapid shifts in the cyanobacterial population and function found may also be used as early disturbance indicator in coastal management practices.

Examination of the diurnal patterns of basic cellular processes in the marine nonheterocystous diazotrophic cyanobacterium Trichodesmium revealed that the division of cells occurred throughout the diurnal cycle, but that it oscillated and peaked at an early stage in the dark period. Transcription of the early cell division gene ftsZ and the occurrence of the FtsZ protein showed a similar diurnal rhythmicity that preceded the division of cells. DNA replication (dnaA gene transcription) occurred before the transcription of ftsZ and hetR, the latter encoding the key heterocyst differentiation protein. Transcription of ftsZ and hetR in turn preceded the development of the nitrogen-fixing diazocytes and nifH transcription, and were at the minimum when diazotrophy was at the maximum. The nifH gene transcription showed a negative correlation to the circadian clock gene kaiC. Together, the data show a temporal separation between cell division and diazotrophy on a diurnal basis.

We analyzed flavodoxin gene sequences and transcription patterns in the marine nitrogen-fixing cyanobacterium Trichodesmium Ehrenberg ex Gomont 1892. While a typical cyanobacterial flavodoxin ortholog ( fld1 ) was identified from cultured (strain IMS101) and environmental Trichodesmium by PCR and Southern blot hybridization, a second flavodoxin ( fld2 ) was identified in the genome sequence. BLAST and phylogenetic analyses indicated that the two fld genes were highly divergent: fld1 was most closely related to fld common in cyanobacteria, wheras fld2 formed a distinct cluster with a fld so far only found in the unicellular diazotrophic cyanobacteria Cyanothece and Crocosphaera (in which fld2 was the only fld ). The fld2 cluster was more closely allied with a fld in non-cyanobacterial diazotrophs such as Azotobacter and Azoarcus than fld1 in other cyanobacteria. This result suggests different evolutionary history of these two genes. Real-Time RT–PCR analysis on iron-replete and iron-deplete Trichodesmium cultures revealed remarkable diel dynamics in transcription of both fld s but surprisingly failed to show induction under iron stress. Addition of nitrate to these cultures depressed nifH and fld2 transcription while elevating the transcription of fld1 in iron-stressed cultures. A positive correlation between fld and nifH transcript abundances was found, which was stronger in the case of fld2 than fld1 . Our data suggest that these two flavodoxins in Trichodesmium may be functionally diversified and that use of flavodoxin as an iron stress indicator needs to be validated for each organism with consideration of diel dynamics of gene expression.

The nitrogen-fixing symbiosis between cyanobacteria and the water fern Azolla microphylla is, in contrast to other cyanobacteria-plant symbioses, the only one of a perpetual nature. The cyanobacterium is vertically transmitted between the plant generations, via vegetative fragmentation of the host or sexually within megasporocarps. In the latter process, subsets of the cyanobacterial population living endophytically in the Azolla leaves function as inocula for the new plant generations. Using electron microscopy and immunogold-labeling, the fate of the cyanobacterium during colonization and development of the megasporocarp was revealed. On entering the indusium chamber of the megasporocarps as small-celled motile cyanobacterial filaments (hormogonia), these differentiated into large thick-walled akinetes (spores) in a synchronized manner. This process was accompanied by cytoplasmic reorganizations and the release of numerous membrane vesicles, most of which contained DNA, and the formation of a highly structured biofilm. Taken together the data revealed complex adaptations in the cyanobacterium during its transition between plant generations.

Annual rates of N2 fixation were measured over 3 yr (1998-2000) at an open water station (BY31) and 2 coastal stations (H4 and X1) in the Baltic Sea. This is the first report on depth- integrated rates of N2 fixation from more than one complete growth season in the Baltic Sea. Annual estimates of N2 fixation ranged from 56 000 to 125 000 t N in the Baltic Proper, and 18 000 to 162 000 t N at the inshore stations (Himmerfjarden). Rates of N2 fixation were measured in situ at 4 depths between 0 and 25 m using the 15 N tracer technique for size fractionated organisms larger and smaller than 20 μm. Maximum rates of N2 fixation were found in surface waters (0 to 4 m depth), and a major part of this activity (80% in coastal and 89% in offshore waters) took place during daylight hours. Integrated rates of N2 fixation in cells > 20 μm followed the average abundance of filamentous cyanobacteria (primarily Aphanizomenon sp.) in the water column. Molar C:N mass ratios in particles >20 μm, i.e. filamentous cyanobacteria, suggest that this size fraction was N-sufficient during sum- mer, whereas the molar C:P mass ratios indicated P-limitation during this period. A reduction in sewage discharge to the Himmerfjarden bay area during the study period appears not to have been compensated for by increased rates of N2 fixation. The patchy distribution of cyanobacteria and the high seasonal variability in N2 fixation rates emphasize the need for adequate spatial and temporal sampling strategies in studies of N2 fixation in coastal and open waters of the Baltic Sea.

Variability in benthic diazotrophy and cyanobacterial diversity in a tropical intertidal lagoon.

Cyanobacteria are now recognized as the primary nitrogen-fixing (diazotrophic) microorganisms in the oceans as contributing significantly to the biogeochemistry of the global nitrogen cycle. The evolution of a remarkable range of morphologies and metabolic capacities has led to a behavioural versatility that helps them to proliferate and combat nutrient limitations in oceanic ecosystems. Advancement in community gene, genome and metagenome analyses of marine microbial communities will further advance functional interpretations and biotechnological uses. In this review we highlight the diversity, adaptation and function of cyanobacteria in the ocean.

Cyanobacteria are highly versatile organisms in spite of their small size; they represent the smallest photosynthetic units on Earth, and gave rise to algae and plants. Cyanobacteria are globally widespread in terrestrial and aquatic environments, including the large oceans. In recent years they have attracted attention because of their high evolutionary importance, their developmental complexity, their unexpected pivotal ecological significance in global biogeochemical cycles and their production of a large number of secondary metabolites, many with great biotechnological potential. In this overview, focus is on the role of cyanobacteria in the origin and evolution of algae and plants, and on mechanisms in contemporary cyanobacterial-plant symbioses (Gunnera and Azolla) that may represent ‘blue-prints’ of those that some 2.1 billion years ago became the first chloroplasts.

The non-protein amino acid beta-N-methyl-amino-L-alanine (BMAA) is a neurotoxin that was recently found to be produced by most cyanobacteria. The neurotoxin was discovered in 1967 in the seeds of the cycad Cycas micronesica, but this BMAA may originate from the symbiotic cyanobacterium Nostoc, which inhabits the roots of cycads. BMAA is thought to be the cause of the deadly neurodegenerative disease amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC), common among the Chamorro people of Guam. It was demonstrated that the Chamorros, in all probability, have been exposed to high levels of BMAA through dietary consumption of flying foxes which fed mainly on cycads seeds. BMAA production may be a common conserved evolutionary feature among cyanobacteria and due to their wide global distribution, the toxin may be a common concern and potentially involved in provoking degenerative diseases worldwide. BMAA may likewise be bioaccumulated in other cyanobacterial based food webs within ecosystems outside Guam, and it is proposed that such webs may exist in the Baltic Sea, with its massive occurrence of cyanobacteria (blooms).

Cyanobacterial symbioses with eukaryotes are ancient associations that are widely distributed in aquatic and terrestrial environments. Cyanobacteria are a significant driving force in the evolution of their hosts, providing a range of services including photosynthesis, nitrogen fixation, UV protection, and defensive toxins. Although widespread, cyanobacteria occur in a limited range of hosts. Terrestrial symbioses are typically restricted to lichens and early evolved plants, and aquatic symbioses to sessile or slow-moving organisms. This review examines the underlying evolutionary processes that may have lead to these patterns. It also examines the facts that the degree of integration between symbiont and host, and the mode of transmission of the symbiont, do not appear to be an indication of how old the symbiosis is or how important it is to host well-being. Biparental transmission of symbionts may prolong the survival of gametes that persist in the environment, increasing chances of fertilization.

Prokaryotic epiphytes on leaves of three seagrass species, Thalassodendron ciliatum, Thalassia hemprichii, and Cymodocea rotundata, from two Kenyan coastal sites, Nyali (a high-nutrient site) and Vipingo (a low-nutrient site), were characterized genetically and morphologically. Denaturing gradient gel electrophoresis (DGGE) and clone libraries of PCR-amplified 16S rRNA gene fragments were used to study prokaryotes associated with these seagrasses. In general, the epiphytic coverage was greater in the high-nutrient site, while the microbial diversity was linked to seagrass species rather than the study sites. Cytophaga–Flavobacteria–Bacteroides (CFB) were associated with T. ciliatum and T. hemprichii mainly in the nutrient-poor site, while α-, β-, and γ-proteobacteria were associated with all three species at the two study sites. Some bacteria phylotypes were closely related to sequences of microorganisms previously recovered from wastewaters or other contaminated sources, indicating the influx of land-based wastes into these coastal lagoon ecosystems. The abundance of potential nitrogen (N2)-fixing cyanobacteria on C. rotundata, particularly in the low-nutrient site, suggested that this association may have been acquired to meet N demands. Unicellular cyanobacteria were dominant and associated with C. rotundata and T. hemprichii (with those on T. hemprichii being closely related to cyanobacterial symbiotic species), while T. ciliatum was almost devoid of cyanobacterial associations at the same site (Nyali), which suggests specificity in the cyanobacteria–seagrass associations. The abundance of prokaryotic epiphytes was considered to be linked to water depth and tidal exposure.

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification “microbialite” origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya , dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis . The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix , two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga - Flavobacterium - Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock.

The filamentous cyanobacteria of the genus Nostoc are globally distributed, phenotypically complex organisms, capable of cellular differentiation and of forming symbiotic associations with a wide range of plants. To further our understanding of these processes and functions, the proteome of photoautotrophically and diazotrophically grown Nostoc sp. PCC 73102 (N. punctiforme) cells was examined. Extracted proteins were separated into membrane and soluble protein fractions and analysed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The analysis led to the identification of 82 proteins that could be divided into 12 functional categories. Significantly, 65 of these proteins have not been previously documented in the Nostoc proteome. Many of the proteins identified were readily recognized as housekeeping proteins involved in carbon, nitrogen and energy metabolism, but a number of proteins related to stress, motility, secretion and post-translational modifications were also identified. Ten unclassified proteins were also detected, representing potential novel functions. These proteins were highly expressed, suggesting that they play key roles during photoautotrophic and diazotrophic growth. Nineteen of the proteins expressed under the growth conditions examined contained putative thioredoxin (Trx) targets, a motif that functions in redox regulation via redox equivalent mediators and is known to be significant in a wide range of biological processes. These observations contribute to our understanding of the complex Nostoc life cycle.

The cellular and subcellular localization of FtsZ, a bacterial cell division protein, were investigated in vegetative cells of the filamentous cyanobacterium Nostoc/Anabaena sp. strain PCC 7120. We show by using immunogold-transmission electron microscopy that FtsZ forms a ring structure in a filamentous cyanobacterium, similar to observations in unicellular bacteria and chloroplasts. This finding, that the FtsZ in a filamentous cyanobacterium accumulates at the growing edge of the division septa leading to the formation of the typical prokaryotic Z-ring arrangement, is novel. Moreover, an apparent cytoplasmic distribution of FtsZ occurred in vegetative cells. During the transition of vegetative cells into terminally differentiated heterocysts the cytoplasmic FtsZ levels decreased substantially. These findings suggest a conserved function of FtsZ among prokaryotes, including filamentous cyanobacteria with cell differentiation capacity, and possibly a role of FtsZ as a cytoskeletal component in the cytoplasm.

Cylindrospermopsis raciborskii CS-505 is an invasive freshwater filamentous cyanobacterium that when grown diazotrophically may develop trichomes of up to 100 vegetative cells while differentiating only two end heterocysts, the sole sites for their N2-fixation process. We examined the diazotrophic growth and intercellular transfer mechanisms in C. raciborskii CS-505. Subjecting cultures to a combined-nitrogen-free medium to elicit N2 fixation, the trichome length remained unaffected while growth rates decreased. The structures and proteins for intercellular communication showed that while a continuous periplasmic space was apparent along the trichomes, the putative septal junction sepJ gene is divided into two open reading frames and lacks several transmembrane domains unlike the situation in Anabaena , differentiating a 5-fold higher frequency of heterocysts. FRAP analyses also showed that the dyes calcein and 5-CFDA were taken up by heterocysts and vegetative cells, and that the transfer from heterocysts and ‘terminal’ vegetative cells showed considerably higher transfer rates than that from vegetative cells located in the middle of the trichomes. The data suggest that C. raciborskii CS-505 compensates its low-frequency heterocyst phenotype by a highly efficient transfer of the fixed nitrogen towards cells in distal parts of the trichomes (growing rapidly) while cells in central parts suffers (slow growth).

Amyotrophic lateral sclerosis (ALS) is an extremely devastating neurodegenerative disease with an obscure etiology. The amino acid β-N-methylamino-l-alanine (BMAA) produced by globally widespread phytoplankton has been implicated in the etiology of human motor neuron diseases [corrected]. BMAA was recently proven to be present in Baltic Sea food webs, ranging from plankton to larger Baltic Sea organisms, some serving as important food items (fish) for humans. To test whether exposure to BMAA in a Baltic Sea setting is reflected in humans, blood and cerebrospinal fluid (CSF) from individuals suffering from ALS were analyzed, together with sex- and age-matched individuals not inflicted with ALS. Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and multiple reaction monitoring (MRM), in conjunction with diagnostic transitions revealed BMAA in three (12%) of the totally 25 Swedish individuals tested, with no preference for those suffering from ALS. The three BMAA-positive samples were all retrieved from the CSF, while BMAA was not detected in the blood. The data show that BMAA, potentially originating from Baltic Sea phytoplankton, may reach the human central nervous system, but does not lend support to the notion that BMAA is resident specifically in ALS-patients. However, while dietary exposure to BMAA may be intermittent and, if so, difficult to detect, our data provide the first demonstration of BMAA in the central nervous system of human individuals ante mortem quantified with UHPLC-MS/MS, and therefore calls for extended research efforts.

Background Insertion sequences (ISs) are approximately 1 kbp long “jumping” genes found in prokaryotes. ISs encode the protein Transposase, which facilitates the excision and reinsertion of ISs in genomes, making these sequences a type of class I (“cut-and-paste”) Mobile Genetic Elements. ISs are proposed to be involved in the reductive evolution of symbiotic prokaryotes. Our previous sequencing of the genome of the cyanobacterium ‘Nostoc azollae’ 0708, living in a tight perpetual symbiotic association with a plant (the water fern Azolla), revealed the presence of an eroding genome, with a high number of insertion sequences (ISs) together with an unprecedented large proportion of pseudogenes. To investigate the role of ISs in the reductive evolution of ‘Nostoc azollae’ 0708, and potentially in the formation of pseudogenes, a bioinformatic investigation of the IS identities and positions in 47 cyanobacterial genomes was conducted. To widen the scope, the IS contents were analysed qualitatively and quantitatively in 20 other genomes representing both free-living and symbiotic bacteria. Results Insertion Sequences were not randomly distributed in the bacterial genomes and were found to transpose short distances from their original location (“local hopping”) and pseudogenes were enriched in the vicinity of IS elements. In general, symbiotic organisms showed higher densities of IS elements and pseudogenes than non-symbiotic bacteria. A total of 1108 distinct repeated sequences over 500 bp were identified in the 67 genomes investigated. In the genome of ‘Nostoc azollae’ 0708, IS elements were apparent at 970 locations (14.3%), with 428 being full-length. Morphologically complex cyanobacteria with large genomes showed higher frequencies of IS elements, irrespective of life style. Conclusions The apparent co-location of IS elements and pseudogenes found in prokaryotic genomes implies earlier IS transpositions into genes. As transpositions tend to be local rather than genome wide this likely explains the proximity between IS elements and pseudogenes. These findings suggest that ISs facilitate the reductive evolution in for instance in the symbiotic cyanobacterium ‘Nostoc azollae’ 0708 and in other obligate prokaryotic symbionts. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1386-7) contains supplementary material, which is available to authorized users.