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Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the β-lactamase–like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P)H:oxygen oxidoreductase, donating electrons directly to O ₂ without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Δ flv1(A) and/or Δ flv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis.

Anabaena variabilis is a diazotrophic filamentous cyanobacterium that differentiates to heterocysts and produces hydrogen as a byproduct. Study on metabolic interactions of the two differentiated cells provides a better understanding of its metabolism especially for improving hydrogen production. To this end, a genome-scale metabolic model for Anabaena variabilis ATCC 29413, iAM957, was reconstructed and evaluated in this research. Then, the model and transcriptomic data of the vegetative and heterocyst cells were applied to construct a regulated two-cell metabolic model. The regulated model improved prediction for biomass in high radiation levels. The regulated model predicts that heterocysts provide an oxygen-free environment and then, this model was used to find strategies for improving hydrogen production in heterocysts. The predictions indicate that the removal of uptake hydrogenase improves hydrogen production which is consistent with previous empirical research. Furthermore, the regulated model proposed activation of some reactions to provide redox cofactors which are required for improving hydrogen production up to 60% by bidirectional hydrogenase.

Nitrogen assimilation is strictly regulated in cyanobacteria. In an inorganic nitrogen-deficient environment, some vegetative cells of the cyanobacterium Anabaena differentiate into heterocysts. We assessed the photosynthesis and nitrogen-fixing capacities of heterocysts and vegetative cells, respectively, at the transcriptome level. RNA extracted from nitrogen-replete vegetative cells (NVs), nitrogen-deprived vegetative cells (NDVs), and nitrogen-deprived heterocysts (NDHs) in Anabaena sp. strain PCC 7120 was evaluated by transcriptome sequencing. Paired comparisons of NVs vs. NDHs, NVs vs. NDVs, and NDVs vs. NDHs revealed 2,044 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs showed that carbon fixation in photosynthetic organisms and several nitrogen metabolism-related pathways were significantly enriched. Synthesis of Gvp (Gas vesicle synthesis protein gene) in NVs was blocked by nitrogen deprivation, which may cause Anabaena cells to sink and promote nitrogen fixation under anaerobic conditions; in contrast, heterocysts may perform photosynthesis under nitrogen deprivation conditions, whereas the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Immunofluorescence analysis of nitrogenase iron protein suggested that the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Our findings provide insight into the molecular mechanisms underlying nitrogen fixation and photosynthesis in vegetative cells and heterocysts at the transcriptome level. This study provides a foundation for further functional verification of heterocyst growth, differentiation, and water bloom control.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous second messenger in bacteria and regulates a variety of cell activities. Many bacteria contain multiple enzymes involved in c-di-GMP synthesis or degradation; however, how they coordinate with each other to orchestrate c-di-GMP homeostasis remains unclear. Here, using the cyanobacterium Anabaena PCC 7120 as a model, we created cdG 0 and cdG max strains by deleting all 8 and 14 genes, respectively, that encode enzymes with c-di-GMP degradation and synthesis domains, alongside a collection of mutants with various numbers of these genes deleted. Our findings demonstrate that c-di-GMP in Anabaena not only modulates cell size but is also indispensable for cell viability. Quantitative analysis established two critical physiological thresholds in vivo: a minimal c-di-GMP level required for cell size maintenance and a lower, lethal threshold essential for survival. We show that the 16 enzymes involved in c-di-GMP turnover in Anabaena function as an electromechanical-like dual relay to control c-di-GMP dynamics, with different modules contributing to c-di-GMP homeostasis or responding as an SOS alarm when the c-di-GMP concentration drops below the lethal threshold. Both effects of c-di-GMP on cell size reduction and cell viability are mediated by the cyclic-di-GMP receptor (CdgR), depending on the amount of the c-di-GMP-free form of CdgR available because of titration by c-di-GMP in the cells. The system, with the two concentration thresholds of c-di-GMP that dictate cell size and viability, respectively, enables dynamic cellular adaptation while preventing lethal effects.

The fixation of atmospheric N2 by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N2 fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N2 fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF+ motif, a palindrome at or close to position –35 that seems essential for heterocyst-specific expression of certain genes.

Cyanobacteria are photosynthetic prokaryotes found in a range of environments. They are infamous for the production of toxins, as well as bioactive compounds, which exhibit anticancer, antimicrobial and protease inhibition activities. Cyanobacteria produce a broad range of antifungals belonging to structural classes, such as peptides, polyketides and alkaloids. Here, we tested cyanobacteria from a wide variety of environments for antifungal activity. The potent antifungal macrolide scytophycin was detected in Anabaena sp. HAN21/1, Anabaena cf. cylindrica PH133, Nostoc sp. HAN11/1 and Scytonema sp. HAN3/2. To our knowledge, this is the first description of Anabaena strains that produce scytophycins. We detected antifungal glycolipopeptide hassallidin production in Anabaena spp. BIR JV1 and HAN7/1 and in Nostoc spp. 6sf Calc and CENA 219. These strains were isolated from brackish and freshwater samples collected in Brazil, the Czech Republic and Finland. In addition, three cyanobacterial strains, Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3, produced unidentified antifungal compounds that warrant further characterization. Interestingly, all of the strains shown to produce antifungal compounds in this study belong to Nostocales or Stigonematales cyanobacterial orders.

Benthic algae fuel summer food webs in many sunlit rivers, and are hotspots for primary and secondary production and biogeochemical cycling. Concerningly, riverine benthic algal assemblages can become dominated by toxic cyanobacteria, threatening water quality and public health. In the Eel River in Northern California, over a dozen dog deaths have been attributed to cyanotoxin poisonings since 2000. During the summers of 2013-2015, we documented spatial and temporal patterns of cyanotoxin concentrations in the watershed, showing widespread distribution of anatoxin-a in benthic cyanobacterial mats. Solid phase adsorption toxin tracking (SPATT) samplers were deployed weekly to record dissolved microcystin and anatoxin-a levels at 10 sites throughout the watershed, and 187 Anabaena-dominated or Phormidium-dominated cyanobacterial mat samples were collected from 27 locations to measure intracellular anatoxin-a (ATX) and microcystins (MCY). Anatoxin-a levels were higher than microcystin for both SPATT (mean MCY = 0.8 and ATX = 4.8 ng g resin-1 day-1) and cyanobacterial mat samples (mean MCY = 0.074 and ATX = 1.89 μg g-1 DW). Of the benthic mats sampled, 58.9% had detectable anatoxin-a (max = 70.93 μg g-1 DW), while 37.6% had detectable microcystins (max = 2.29 μg g-1 DW). SPATT cyanotoxin levels peaked in mid-summer in warm mainstem reaches of the watershed. This is one of the first documentations of widespread anatoxin-a occurrence in benthic cyanobacterial mats in a North American watershed.

Soil salinity in nature is generally mixed type; however, most of the studies on salt toxicity are performed with NaCl and little is known about sulfur type of salinity (Na2SO4). Present study discerns the physiologic mechanisms responsible for salt tolerance in salt-adapted Anabaena fertilissima, and responses of directly stressed parent cells to NaCl and NaCl+Na2SO4 mixture. NaCl at 500 mM was lethal to the cyanobacterium, whereas salt-adapted cells grew luxuriantly. Salinity impaired gross photosynthesis, electron transport activities, and respiration in parent cells, but not in the salt-adapted cells, except a marginal increase in PSI activity. Despite higher Na+ concentration in the salt mixture, equimolar NaCl appeared more inhibitive to growth. Sucrose and trehalose content and antioxidant activities were maximal in 250 mM NaCl-treated cells, followed by salt mixture and was almost identical in salt-adapted (exposed to 500 mm NaCl) and control cells, except a marginal increase in ascorbate peroxidase activity and an additional fourth superoxide dismutase isoform. Catalase isoform of 63 kDa was induced only in salt-stressed cells. Salinity increased the uptake of intracellular Na+ and Ca2+ and leakage of K+ in parent cells, while cation level in salt-adapted cells was comparable to control. Though there was differential increase in intracellular Ca2+ under different salt treatments, ratio of Ca2+/Na+ remained the same. It is inferred that stepwise increment in the salt concentration enabled the cyanobacterium to undergo priming effect and acquire robust and efficient defense system involving the least energy.

Under nitrogen deprivation, the one-dimensional cyanobacterial organism Anabaena sp. PCC 7120 develops patterns of single, nitrogen-fixing cells separated by nearly regular intervals of photosynthetic vegetative cells. We study a minimal, stochastic model of developmental patterns in Anabaena that includes a nondiffusing activator, two diffusing inhibitor morphogens, demographic fluctuations in the number of morphogen molecules, and filament growth. By tracking developing filaments, we provide experimental evidence for different spatiotemporal roles of the two inhibitors during pattern maintenance and for small molecular copy numbers, justifying a stochastic approach. In the deterministic limit, the model yields Turing patterns within a region of parameter space that shrinks markedly as the inhibitor diffusivities become equal. Transient, noise-driven, stochastic Turing patterns are produced outside this region, which can then be fixed by downstream genetic commitment pathways, dramatically enhancing the robustness of pattern formation, also in the biologically relevant situation in which the inhibitors' diffusivities may be comparable.

Hexachlorocyclohexane (HCH) was extensively used as a pesticide until the 1990s. It was synthesized by benzene photochlorination, resulting in a mixture of stereoisomers, which included α‐, β‐, γ‐, and δ‐HCH, among others. It was later discovered that only the γ‐HCH isomer (also called lindane) had insecticidal properties, so it began to be purified from this mixture, while the remaining HCH isomers (representing around 85%–90% of industrial HCH production) were disposed of in dumpsites, generating environmental issues. Several works have studied microbial‐driven biodegradation and physiological responses to γ‐HCH, but information concerning the other isomers is scarce. Since previous research showed that the cyanobacterium Anabaena sp. PCC 7120 is effective at removing lindane; this study focused on its responses to the α‐, β‐, and δ‐HCH isomers. The results showed that Anabaena tolerates α‐ and γ‐HCH well, with little impact on growth, while β‐ and δ‐HCH are more poorly tolerated and negatively affect growth and cell physiology. It was also found that, in the presence of Anabaena sp. PCC 7120, both α‐ and γ‐HCH are completely eliminated from supernatants while β‐ and δ‐HCH are partially eliminated. Additionally, the linC gene was found to be expressed at twice the normal level in the presence of α‐ and γ‐HCH at 2 mg/mL. Overall, this study reveals how Anabaena responds to key HCH isomers found in contaminated sites and supports its potential use in bioremediation. This study investigates the physiological responses of Anabaena sp. PCC 7120 to HCH isomers, emphasizing both its tolerance to these compounds and its ability to biodegrade them. The findings support the potential application of Anabaena in the bioremediation of environments contaminated with specific HCH isomers.