Long-term stability of the genome structure of the cyanobacterium, Dolichospermum in a deep German lake.

Dolichospermum is a cyanobacterial genus commonly associated with toxic blooms in lakes and brackish water bodies worldwide, and is a long-term resident of Lake Stechlin, northeastern Germany. In recent decades, shifts in the phosphorus loading and phytoplankton species composition have seen increased biomass of Dolichospermum during summer blooms from 1998, peaking around 2005, and declining after 2020. Cyanobacteria are known to rapidly adapt to new environments, facilitated by genome adaptation. To investigate the changes in genomic features that may have occurred in Lake Stechlin Dolichospermum during this time of increased phosphorus loading and higher biomass, whole genome sequence analysis was performed on samples of ten akinetes isolated from ten, 1 cm segments of a sediment core, representing a ∼45-year period from 1970 to 2017. Comparison of these genomes with genomes of extant isolates revealed a clade of Dolichospermum that clustered with the ADA-6 genus complex, with remarkable genome stability, without gene gain or loss events in response to recent environmental changes. The genome characteristics indicate that this species is suited to a deep-chlorophyll maximum, including additional light-harvesting and phosphorus scavenging genes. Population SNP analysis revealed two sub-populations that shifted in dominance as the lake transitioned between oligotrophic and eutrophic conditions. Overall, the results show little change within the population, despite diversity between extant populations from different geographic locations and the in-lake changes in phosphorus concentrations.

Changes in Envelope Structure and Cell-Cell Communication during Akinete Differentiation and Germination in Filamentous Cyanobacterium Trichormus variabilis ATCC 29413.

Planktonic freshwater filamentous cyanobacterium Trichormus variabilis ATCC 29413 (previously known as Anabaena variabilis) can differentiate heterocysts and akinetes to survive under different stress conditions. Whilst heterocysts enable diazotrophic growth, akinetes are spore-like resting cells that make the survival of the species possible under adverse growth conditions. Under suitable environmental conditions, they germinate to produce new vegetative filaments. Several morphological and physiological changes occur during akinete formation and germination. Here, using scanning electron microscopy (SEM), we found that the mature akinetes had a wrinkled envelope, and the surface of the envelope smoothened as the cell size increased during germination. Thereupon, the akinete envelope ruptured to release the short emerging filament. Focused ion beam-scanning electron microscopy (FIB/SEM) tomography of immature akinetes revealed the presence of cytoplasmic granules, presumably consisting of cyanophycin or glycogen. In addition, the akinete envelope architecture of different layers, the exopolysaccharide and glycolipid layers, could be visualized. We found that this multilayered envelope helped to withstand osmotic stress and to maintain the structural integrity. Furthermore, by fluorescence recovery after photobleaching (FRAP) measurements, using the fluorescent tracer calcein, we found that intercellular communication decreased during akinete formation as compared with the vegetative cells. In contrast, freshly germinating filaments restored cell communication.

Akinetes From Late Paleoproterozoic Salkhan Limestone (>1600 Ma) of India: A Proxy for Understanding Life in Extreme Conditions.

Isolated elongate spore-like cells present in the >1600 Ma-old Salkhan Limestone of the Semri Group, Vindhayan Supergroup, India are considered akinetes of the heterocystous cyanobacteria. Small to large size, and young (single walled) to mature (double walled) akinetes - namely, Archaeoellipsoides bactroformis, A. conjuctivus, A. dolichos, A. elongatus, A. grandis, A. major and A. minor - found in the stromatolitic and bedded cherts have been reported in the present paper. Their role in understanding extreme environmental conditions is a subject matter of this paper. Additionally, the occurrence of doubly-terminated quartz crystals and fan-fabrics in the Salkhan Limestone indicates adverse conditions for the survival of life forms. The depositional environment of the Salkhan Limestone, Vindhyan Supergroup is suggested to be shallow marine intertidal with pulses of the intermittent hypersaline regime during which akinetes, closely resembling those of extant Nostocaceans, were formed by cyanobacteria for survival in the extreme conditions.

A highly asynchronous developmental program triggered during germination of dormant akinetes of filamentous diazotrophic cyanobacteria.

Germination of akinetes of filamentous heterocyst-forming cyanobacteria of the order Nostocales is an essential process that ensures survival and recolonization after long periods of unfavorable conditions, as desiccation, cold and low light. We studied the morphological, physiological and metabolic changes that occur during germination of akinetes in two model species of cell differentiation, Anabaena variabilis ATCC 29413 and Nostoc punctiforme ATCC 29133, which live in different habitats. We characterized the akinete envelopes and showed their similarity to envelopes of N2-fixing heterocysts. Akinete germination started inside the envelopes and was dependent on light intensity but independent of nitrogen supply. During the germination of A. variabilis akinetes, cell division and heterocyst differentiation were highly accelerated. The energy for cell division was initially supplied by respiration of glycogen and subsequently by photosynthesis. By contrast, during germination of N. punctiforme akinetes, cell division and heterocyst differentiation were slow. During the initial 15-20 h, N. punctiforme akinetes increased in volume and some burst. Only then did intact akinetes start to divide and fully germinate, possibly fueled by nutrients released from dead akinetes. The different strategies used by these different cyanobacteria allow successful germination of dormant cells and recolonization under favorable conditions.

The Presence of Toxic and Non-Toxic Cyanobacteria in the Sediments of the Limpopo River Basin: Implications for Human Health.

The presence of harmful algal blooms (HABs) and cyanotoxins in drinking water sources poses a great threat to human health. The current study employed molecular techniques to determine the occurrence of non-toxic and toxic cyanobacteria species in the Limpopo River basin based on the phylogenetic analysis of the 16S rRNA gene. Bottom sediment samples were collected from selected rivers: Limpopo, Crocodile, Mokolo, Mogalakwena, Nzhelele, Lephalale, Sand Rivers (South Africa); Notwane (Botswana); and Shashe River and Mzingwane River (Zimbabwe). A physical-chemical analysis of the bottom sediments showed the availability of nutrients, nitrates and phosphates, in excess of 0.5 mg/L, in most of the river sediments, while alkalinity, pH and salinity were in excess of 500 mg/L. The FlowCam showed the dominant cyanobacteria species that were identified from the sediment samples, and these were the Microcystis species, followed by Raphidiopsis raciborskii, Phormidium and Planktothrix species. The latter species were also confirmed by molecular techniques. Nevertheless, two samples showed an amplification of the cylindrospermopsin polyketide synthetase gene (S3 and S9), while the other two samples showed an amplification for the microcystin/nodularin synthetase genes (S8 and S13). Thus, these findings may imply the presence of toxic cyanobacteria species in the studied river sediments. The presence of cyanobacteria may be hazardous to humans because rural communities and farmers abstract water from the Limpopo river catchment for human consumption, livestock and wildlife watering and irrigation.

Benthic Archives Reveal Recurrence and Dominance of Toxigenic Cyanobacteria in a Eutrophic Lake over the Last 220 Years.

Akinetes are resistant cells which have the ability to persist in sediment for several decades. We have investigated the temporal distribution of akinetes of two species, Dolichospermum macrosporum and Dolichospermum flos-aquae, in a sediment core sampled in Lake Aydat (France), which covers 220 years. The upper part, from 1907 to 2016, the number of akinetes fluctuated but stayed at high concentrations, especially for D. macrosporum in surface sediment (with the maximal value close to 6.105 akinetes g DW-1 of sediment), suggesting a recurrence of blooms of this species which was probably closely related to anthropic eutrophication since the 1960s. Before 1907, the abundance of akinetes of both species was very low, suggesting only a modest presence of these cyanobacteria. In addition, the percentage of intact akinetes was different for each species, suggesting different ecological processes in the water column. This percentage also decreased with depth, revealing a reduction in germination potential over time. In addition, biosynthetic genes of anatoxin-a (anaC) and microcystin (mcyA) were detected. First results show a high occurrence of mcyA all down the core. In contrast, anaC gene was mostly detected in the surface sediment (since the 1980s), revealing a potentially more recent occurrence of this cyanotoxin in Lake Aydat which may be associated with the recurrence of blooms of D. macrosporum and thus with anthropic activities.

Evaluating methods for purifying cyanobacterial cultures by qPCR and high-throughput Illumina sequencing.

Cyanobacteria are commonly found in association with other microorganisms, which constitutes a great challenge during the isolation of cyanobacterial strains. Although several methods have been published for obtaining axenic cyanobacterial cultures, their efficiency is usually evaluated by observing the growth of non-cyanobacteria in culture media. In order to verify whether uncultured bacteria should be a concern during cyanobacterial isolation, this work aimed to detect by molecular methods sequences from cyanobacteria and other bacteria present before and after a technique for obtaining axenic cultures from plating and exposure of Fischerella sp. CENA161 akinetes to the Extran detergent and sodium hypochlorite. Solutions containing 0.5, 1, and 2% sodium hypochlorite were able to remove contaminant bacterial CFUs from the culture. However, qPCR pointed that the quantity of sequences amplified with universal bacteria primers was higher than the number of cyanobacteria-specific sequences before and after treatments. The presence of uncultured bacteria in post-hypochlorite cultures was confirmed by high-throughput Illumina sequencing. These results suggest that culturing may overlook the presence of uncultured bacteria associated to cyanobacterial strains and is not sufficient for monitoring the success of cyanobacterial isolation by itself. Molecular methods such as qPCR could be employed as an additional measure for evaluating axenity in cyanobacterial strains.

Characterization of akinetes from cyanobacterial strains and lake sediment: A study of their resistance and toxic potential.

Nostocalean cyanobacteria are known to proliferate abundantly in eutrophic aquatic ecosystems, and to produce several cyanotoxins, including anatoxin-a. In this study, we investigated both the resistance and toxic potential of the akinetes (resistant cells), using cyanobacterial cultures and akinetes extracted from the sediment of Lake Aydat (France) sampled in the winter and spring. Intact and lysed akinetes were differentiated using a double control based on the autofluorescence of akinetes and SYTOX-green staining. The percentage of resistant akinetes found in several different abiotic stress conditions was highly variable, depending on the species and also on the sampling season. Thus, the resistance of akinetes and their ability to germinate seems to follow a species-specific process, and akinetes can undergo physiologic changes during the sedimentary phase of the Nostocale life cycle. This study also revealed the first evidence of anatoxin-a genes in akinetes, with anaC and anaF genes detected in akinetes from all cyanobacterial producer cultures. The low number of anaC genes, almost exclusively detected using nested PCR, in the sediment at Lake Aydat suggests a limited but existent past population of toxic Nostocales in this lake. Given the key role of akinetes in the annual cycle and subsequent summer proliferation, it can be interesting to integrate the surveillance of akinetes in the management of lakes exposed to recurrent cyanobacterial blooms.

Carbon assimilation and accumulation of cyanophycin during the development of dormant cells (akinetes) in the cyanobacterium Aphanizomenon ovalisporum.

Akinetes are spore-like non-motile cells that differentiate from vegetative cells of filamentous cyanobacteria from the order Nostocales. They play a key role in the survival and distribution of these species and contribute to their perennial blooms. Here, we demonstrate variations in cellular ultrastructure during akinete formation concomitant with accumulation of cyanophycin; a copolymer of aspartate and arginine that forms storage granules. Cyanophycin accumulation is initiated in vegetative cells few days post-exposure to akinete inducing conditions. This early accumulated cyanophycin pool in vegetative cells disappears as a nearby cell differentiates to an akinete and stores large pool of cyanophycin. During the akinete maturation, the cyanophycin pool is further increased and comprise up to 2% of the akinete volume. The cellular pattern of photosynthetic activity during akinete formation was studied by a nano-metric scale secondary ion mass spectrometry (NanoSIMS) analysis in (13)C-enriched cultures. Quantitative estimation of carbon assimilation in vegetative cells and akinetes (filament-attached and -free) indicates that vegetative cells maintain their basal activity while differentiating akinetes gradually reduce their activity. Mature-free akinetes practically lost their photosynthetic activity although small fraction of free akinetes were still photosynthetically active. Additional (13)C pulse-chase experiments indicated rapid carbon turnover during akinete formation and de novo synthesis of cyanophycin in vegetative cells 4 days post-induction of akinete differentiation.

Immunocytochemical localization of Fe-SOD in different cells of Anabaena cylindrica Lemm. grown at two different photon irradiances.

Superoxide dismutase (SOD) converts superoxide radicals into hydrogen peroxide and molecular oxygen and therefore represents the primary defence against oxygen damage. Using antibodies against Anabaena cylindrica Lemm. iron-SOD, the isoenzyme was localized by immunogold labelling in vegetative cells, heterocysts and akinetes of the cyanobacterium Anabaena cylindrica grown at different light intensities. At a low photon irradiance (25 µmol photons m-2 s-1 ), the density of iron-SOD labelling was similar in vegetative cells and in heterocysts, whereas in akinetes only 60 % of the gold particles were present as compared with the other cell types. At a higher photon irradiance (140 µmol photons m-2 S-1 ), iron-SOD labelling increased by 100% in both vegetative cells and in heterocysts but did not change in akinetes. The cultures grown at the photon irradiance of 140 µmol photons m-2 s-1 showed a rate of photosynthetic oxygen evolution 40 % higher than that of cultures grown at the lower photon irradiance. The increase of SOD labelling in vegetative cells is in line with the increased photosynthetic oxygen evolution induced by light. The unchanged SOD labelling observed in the akinetes suggests that light intensity does not seem to affect the oxidative metabolism due to respiratory activity.