Synergistic regulation of hydrogen sulfide and nitric oxide on biochemical components, exopolysaccharides, and nitrogen metabolism in nickel stressed rice field cyanobacteria.

The present study examined the regulatory mechanism of hydrogen sulfide (H2S) and nitric oxide (NO) in nickel (Ni) stressed cyanobacteria viz., Nostoc muscorum and Anabaena sp. by analyzing growth, photosynthetic pigments, biochemical components (protein and carbohydrate), exopolysaccharides (EPS), inorganic nitrogen content, and activity of enzymes comprised in nitrogen metabolism and Ni accumulation. The 1 µM Ni substantially diminished growth by 18% and 22% in N. muscorum and Anabaena sp. respectively, along with declining the pigment contents (Chl a/Car ratio and phycobiliproteins), and biochemical components. It also exerted negative impacts on inorganic uptake of nitrate and nitrite contents; nitrate reductase and nitrite reductase; and ammonium assimilating enzymes (glutamine synthetase, glutamate synthase, and glutamate dehydrogenase exhibited a reverse trend) activities. Nonetheless, the adverse impact of Ni can be mitigated through the exogenous supplementation of NaHS [sodium hydrosulfide (8 µM); H2S donor] and SNP [sodium nitroprusside (10 µM); NO donor] which showed substantial improvement on growth, pigments, nitrogen metabolism, and EPS layer and noticeably occurred as a consequence of a substantial reduction in Ni accumulation content which minimized the toxicity effects. The accumulation of Ni on both the cyanobacterial cell surface (EPS layer) are confirmed by the SEM-EDX analysis. Further, the addition of NO scavenger (PTIO; 20 µM) and inhibitor of NO (L-NAME; 100 µM); and H2S scavenger (HT; 20 µM) and H2S inhibitor (PAG; 50 µM) reversed the positive responses of H2S and NO and damages were more prominent under Ni stress thereby, suggesting the downstream signaling of H2S on NO-mediated alleviation. Thus, this study concludes the crosstalk mechanism of H2S and NO in the mitigation of Ni-induced toxicity in rice field cyanobacteria.

Functional Diversity of TonB-Like Proteins in the Heterocyst-Forming Cyanobacterium Anabaena sp. PCC 7120.

The TonB-dependent transport of scarcely available substrates across the outer membrane is a conserved feature in Gram-negative bacteria. The plasma membrane-embedded TonB-ExbB-ExbD accomplishes complex functions as an energy transducer by physically interacting with TonB-dependent outer membrane transporters (TBDTs). TonB mediates structural rearrangements in the substrate-loaded TBDTs that are required for substrate translocation into the periplasm. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, four TonB-like proteins have been identified. Out of these TonB3 accomplishes the transport of ferric schizokinen, the siderophore which is secreted by Anabaena to scavenge iron. In contrast, TonB1 (SjdR) is exceptionally short and not involved in schizokinen transport. The proposed function of SjdR in peptidoglycan structuring eliminates the protein from the list of TonB proteins in Anabaena. Compared with the well-characterized properties of SjdR and TonB3, the functions of TonB2 and TonB4 are yet unknown. Here, we examined tonB2 and tonB4 mutants for siderophore transport capacities and other specific phenotypic features. Both mutants were not or only slightly affected in schizokinen transport, whereas they showed decreased nitrogenase activity in apparently normal heterocysts. Moreover, the cellular metal concentrations and pigment contents were altered in the mutants, most pronouncedly in the tonB2 mutant. This strain showed an altered susceptibility toward antibiotics and SDS and formed cell aggregates when grown in liquid culture, a phenotype associated with an elevated lipopolysaccharide (LPS) production. Thus, the TonB-like proteins in Anabaena appear to take over distinct functions, and the mutation of TonB2 strongly influences outer membrane integrity. IMPORTANCE The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified. In addition to a role in uptake of scarcely available nutrients, including iron complexes, TonB proteins are related to virulence, flagellum assembly, pilus localization, or envelope integrity, including antibiotic resistance. The knowledge about the function of TonB proteins in cyanobacteria is limited. Here, we compare the four TonB proteins of Anabaena sp. strain PCC 7120, providing evidence that their functions are in part distinct, since mutants of these proteins exhibit specific features but also show some common impairments.

Production of polyhydroxybutyrate by the cyanobacterium cf. Anabaena sp.

Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 gPHB gbiomass-1 d-1) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L-1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L-1, and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions.

A TonB-Like Protein, SjdR, Is Involved in the Structural Definition of the Intercellular Septa in the Heterocyst-Forming Cyanobacterium Anabaena.

Cyanobacteria are photosynthetic organisms with a Gram-negative envelope structure. Certain filamentous species such as Anabaena sp. strain PCC 7120 can fix dinitrogen upon depletion of combined nitrogen. Because the nitrogen-fixing enzyme, nitrogenase, is oxygen sensitive, photosynthesis and nitrogen fixation are spatially separated in Anabaena. Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate from vegetative cells. During heterocyst differentiation, a microoxic environment is created by dismantling photosystem II and restructuring the cell wall. Moreover, solute exchange between the different cell types is regulated to limit oxygen influx into the heterocyst. The septal zone containing nanopores for solute exchange is constricted between heterocysts and vegetative cells, and cyanophycin plugs are located at the heterocyst poles. We identified a protein previously annotated as TonB1 that is largely conserved among cyanobacteria. A mutant of the encoding gene formed heterocysts but was impaired in diazotrophic growth. Mutant heterocysts appeared elongated and exhibited abnormal morphological features, including a reduced cyanophycin plug, an enhanced septum size, and a restricted nanopore zone in the septum. In spite of this, the intercellular transfer velocity of the fluorescent marker calcein was increased in the mutant compared to the wild type. Thus, the protein is required for proper formation of septal structures, expanding our emerging understanding of Anabaena peptidoglycan plasticity and intercellular solute exchange, and is therefore renamed SjdR (septal junction disk regulator). Notably, calcium supplementation compensated for the impaired diazotrophic growth and alterations in septal peptidoglycan in the sjdR mutant, emphasizing the importance of calcium for cell wall structure. IMPORTANCE Multicellularity in bacteria confers an improved adaptive capacity to environmental conditions and stresses. This includes an enhanced capability of resource utilization through a distribution of biochemical processes between constituent cells. This specialization results in a mutual dependency of different cell types, as is the case for nitrogen-fixing heterocysts and photosynthetically active vegetative cells in Anabaena. In this cyanobacterium, intercellular solute exchange is facilitated through nanopores in the peptidoglycan between adjacent cells. To ensure functionality of the specialized cells, septal size as well as the position, size, and frequency of nanopores in the septum need to be tightly established. The novel septal junction disk regulator SjdR characterized here is conserved in the cyanobacterial phylum. It influences septal size and septal nanopore distribution. Consequently, its absence severely affects the intercellular communication and the strains' growth capacity under nitrogen depletion. Thus, SjdR is involved in septal structure remodeling in cyanobacteria.

Comparative Phenotypic Analysis of Anabaena sp. PCC 7120 Mutants of Porinlike Genes.

Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834, alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.

Thioredoxin pathway in Anabaena sp. PCC 7120: activity of NADPH-thioredoxin reductase C.

To understand the physiological role of NADPH-thioredoxin reductase C (NTRC) in cyanobacteria, we investigated an NTRC-deficient mutant strain of Anabaena sp., PCC 7120, cultivated under different regimes of nitrogen supplementation and light exposure. The deletion of ntrC did not induce a change in the cell structure and metabolic pathways. However, time-dependent changes in the abundance of specific proteins and metabolites were observed. A decrease in chlorophyll a was correlated with a decrease in chlorophyll a biosynthesis enzymes and photosystem I subunits. The deletion of ntrC led to a deregulation of nitrogen metabolism, including the NtcA accumulation and heterocyst-specific proteins while nitrate ions were available in the culture medium. Interestingly, this deletion resulted in a redox imbalance, indicated by higher peroxide levels, higher catalase activity and the induction of chaperones such as MsrA. Surprisingly, the antioxidant protein 2-CysPrx was downregulated. The deficiency in ntrC also resulted in the accumulation of metabolites such as 6-phosphogluconate, ADP and ATP. Higher levels of NADP+ and NADPH partly correlated with higher G6PDH activity. Rather than impacting protein expression levels, NTRC appears to be involved in the direct regulation of enzymes, especially during the dark-to-light transition period.

Calm on the surface, dynamic on the inside. Molecular homeostasis of Anabaena sp. PCC 7120 nitrogen metabolism.

Nitrogen sources are all converted into ammonium/ia as a first step of assimilation. It is reasonable to expect that molecular components involved in the transport of ammonium/ia across biological membranes connect with the regulation of both nitrogen and central metabolism. We applied both genetic (i.e., Δamt mutation) and environmental treatments to a target biological system, the cyanobacterium Anabaena sp PCC 7120. The aim was to both perturb nitrogen metabolism and induce multiple inner nitrogen states, respectively, followed by targeted quantification of key proteins, metabolites and enzyme activities. The absence of AMT transporters triggered a substantial whole-system response, affecting enzyme activities and quantity of proteins and metabolites, spanning nitrogen and carbon metabolisms. Moreover, the Δamt strain displayed a molecular fingerprint indicating nitrogen deficiency even under nitrogen replete conditions. Contrasting with such dynamic adaptations was the striking near-complete lack of an externally measurable altered phenotype. We conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. This analysis provides evidence for a potential role of AMT transporters in the regulatory/signalling network of nitrogen metabolism and the existence of a novel fourth regulatory mechanism controlling glutamine synthetase activity.

The Two TpsB-Like Proteins in Anabaena sp. Strain PCC 7120 Are Involved in Secretion of Selected Substrates.

The outer membrane of Gram-negative bacteria acts as an initial diffusion barrier that shields the cell from the environment. It contains many membrane-embedded proteins required for functionality of this system. These proteins serve as solute and lipid transporters or as machines for membrane insertion or secretion of proteins. The genome of Anabaena sp. strain PCC 7120 codes for two outer membrane transporters termed TpsB1 and TpsB2. They belong to the family of the two-partner secretion system proteins which are characteristic of pathogenic bacteria. Because pathogenicity of Anabaena sp. strain PCC 7120 has not been reported, the function of these two cyanobacterial TpsB proteins was analyzed. TpsB1 is encoded by alr1659, while TpsB2 is encoded by all5116 The latter is part of a genomic region containing 11 genes encoding TpsA-like proteins. However, tpsB2 is transcribed independently of a tpsA gene cluster. Bioinformatics analysis revealed the presence of at least 22 genes in Anabaena sp. strain PCC 7120 putatively coding for substrates of the TpsB system, suggesting a rather global function of the two TpsB proteins. Insertion of a plasmid into each of the two genes resulted in altered outer membrane integrity and antibiotic resistance. In addition, the expression of genes coding for the Clp and Deg proteases is dysregulated in these mutants. Moreover, for two of the putative substrates, a dependence of the secretion on functional TpsB proteins could be confirmed. We confirm the existence of a two-partner secretion system in Anabaena sp. strain PCC 7120 and predict a large pool of putative substrates.IMPORTANCE Cyanobacteria are important organisms for the ecosystem, considering their contribution to carbon fixation and oxygen production, while at the same time some species produce compounds that are toxic to their environment. As a consequence, cyanobacterial overpopulation might negatively impact the diversity of natural communities. Thus, a detailed understanding of cyanobacterial interaction with the environment, including other organisms, is required to define their impact on ecosystems. While two-partner secretion systems in pathogenic bacteria are well known, we provide a first description of the cyanobacterial two-partner secretion system.

Phytohormone up-regulates the biochemical constituent, exopolysaccharide and nitrogen metabolism in paddy-field cyanobacteria exposed to chromium stress.

BACKGROUND: Cyanobacteria are well known for their inherent ability to serve as atmospheric nitrogen fixers and as bio-fertilizers; however, increased contaminants in aquatic ecosystem significantly decline the growth and function of these microbes in paddy fields. Plant growth regulators play beneficial role in combating the negative effects induced by heavy metals in photoautotroph. Current study evaluates the potential role of indole acetic acid (IAA; 290 nm) and kinetin (KN; 10 nm) on growth, nitrogen metabolism and biochemical constituents of two paddy field cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 exposed to two concentrations of chromium (CrVI; 100 µM and 150 µM). RESULTS: Both the tested doses of CrVI declined the growth, ratio of chlorophyll a to carotenoids (Chl a/Car), contents of phycobiliproteins; phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE), protein and carbohydrate associated with decrease in the inorganic nitrogen (nitrate; NO3- and nitrite; NO2-) uptake rate that results in the decrease in nitrate and ammonia assimilating enzymes; nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT) except glutamate dehydrogenase (GDH). However, exogenous supplementation of IAA and KN exhibited alleviating effects on growth, nitrogen metabolism and exopolysaccharide (EPS) (first protective barrier against metal toxicity) contents in both the cyanobacteria, which probably occurred as a result of a substantial decrease in the Cr uptake that lowers the damaging effects. CONCLUSION: Overall result of the present study signifies affirmative role of the phytohormone in minimizing the toxic effects induced by chromium by stimulating the growth of cyanobacteria thereby enhancing its ability as bio-fertilizer that improved fertility and productivity of soil even in metal contaminated condition.

Function analysis of RNase E in the filamentous cyanobacterium Anabaena sp. PCC 7120.

RNase E is an endoribonuclease and plays a central role in RNA metabolism. Cyanobacteria, as ancient oxygen-producing photosynthetic bacteria, also contain RNase E homologues. Here, we introduced mutations into the S1 subdomain (F53A), the 5'-sensor subdomain (R160A), and the DNase I subdomain (D296A) according to the key activity sites of Escherichia coli RNase E. The results of degradation assays demonstrated that Asp296 is important to RNase E activity in Anabaena sp. PCC 7120 (hereafter PCC 7120). The docking model of RNase E in PCC 7120 (AnaRne) and RNA suggested a possible recognition mechanism of AnaRne to RNA. Moreover, overexpression of AnaRne and its N-terminal catalytic domain (AnaRneN) in vivo led to the abnormal cell division and inhibited the growth of PCC 7120. The quantitative analysis showed a significant decrease of ftsZ transcription in the case of overexpression of AnaRne or AnaRneN and ftsZ mRNA could be directly degraded by AnaRne through degradation assays in vitro, indicating that AnaRne was related to the expression of ftsZ and eventually affected cell division. In essence, our studies expand the understanding of the structural and functional evolutionary basis of RNase E and lay a foundation for further analysis of RNA metabolism in cyanobacteria.

Conditioning film formation and its influence on the initial adhesion and biofilm formation by a cyanobacterium on photobioreactor materials.

Although cyanobacteria are a common group of microorganisms well-suited to utilization in photobioreactors (PBRs), studies of cyanobacteria fouling and its prevention are scarce. Using a cyanobacterium, Anabaena sp. PCC 7120, which had been genetically modified to enhance linalool production, the formation of conditioning films and the effects of these on the physico-chemical surface properties of various PBR materials during initial adhesion and biofilm formation were investigated. The adhesion assay revealed that the overall attachment of Anabaena was substratum dependent and no correlation between the hydrophobicity/roughness of clean material and cell attachment was found. Surface hydrophilicity/hydrophobicity of all the materials changed within 12 h due to formation of conditioning films. ATR-FTIR spectroscopy revealed that the fractional change in protein deposition between 12 to 96 h was consistent with Anabaena cell attachment but polysaccharide deposition was material specific and did not correlate with cell attachment on the PBR materials. Also, the delay in conditioning film proteins on PVC and PTFE indicated that components other than proteins may be responsible for the decrease in contact angles on these surfaces within 12 h. This indicates the important role of the chemical nature of adsorbed conditioning films in determining the initial attachment of Anabaena to PBR materials. The lower rate of attachment of Anabaena on the hydrophilic surfaces (glass and PMMA) between 72 h to 96 h (regime 3) showed that these surfaces could potentially have low fouling characteristics at extended time scales and should be considered for further research.

The small Ca2+-binding protein CSE links Ca2+ signalling with nitrogen metabolism and filament integrity in Anabaena sp. PCC 7120.

BACKGROUND: Filamentous cyanobacteria represent model organisms for investigating multicellularity. For many species, nitrogen-fixing heterocysts are formed from photosynthetic vegetative cells under nitrogen limitation. Intracellular Ca2+ has been implicated in the highly regulated process of heterocyst differentiation but its role remains unclear. Ca2+ is known to operate more broadly in metabolic signalling in cyanobacteria, although the signalling mechanisms are virtually unknown. A Ca2+-binding protein called the Ca2+ Sensor EF-hand (CSE) is found almost exclusively in filamentous cyanobacteria. Expression of asr1131 encoding the CSE protein in Anabaena sp. PCC 7120 was strongly induced by low CO2 conditions, and rapidly downregulated during nitrogen step-down. A previous study suggests a role for CSE and Ca2+ in regulation of photosynthetic activity in response to changes in carbon and nitrogen availability. RESULTS: In the current study, a mutant Anabaena sp. PCC 7120 strain lacking asr1131 (Δcse) was highly prone to filament fragmentation, leading to a striking phenotype of very short filaments and poor growth under nitrogen-depleted conditions. Transcriptomics analysis under nitrogen-replete conditions revealed that genes involved in heterocyst differentiation and function were downregulated in Δcse, while heterocyst inhibitors were upregulated, compared to the wild-type. CONCLUSIONS: These results indicate that CSE is required for filament integrity and for proper differentiation and function of heterocysts upon changes in the cellular carbon/nitrogen balance. A role for CSE in transmitting Ca2+ signals during the first response to changes in metabolic homeostasis is discussed.

A novel septal protein of multicellular heterocystous cyanobacteria is associated with the divisome.

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.

Effect of growth temperature on biosynthesis and accumulation of carotenoids in cyanobacterium Anabaena sp. PCC 7120 under diazotrophic conditions.

Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) ß-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of ß-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of ß-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between ß-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures.

Disruption of the Gene trx-m1 Impedes the Growth of Anabaena sp. PCC 7120 under Nitrogen Starvation.

Cyanobacteria possess a sophisticated photosynthesis-based metabolism with admirable plasticity. This plasticity is possible via the deep regulation network, the thiol-redox regulations operated by thioredoxin (hereafter, Trx). In this context, we characterized the Trx-m1-deficient mutant strain of Anabaena sp., PCC 7120 (shortly named A.7120), cultivated under nitrogen limitation. Trx-m1 appears to coordinate the nitrogen response and its absence induces large changes in the proteome. Our data clearly indicate that Trx-m1 is crucial for the diazotrophic growth of A.7120. The lack of Trx-m1 resulted in a large differentiation of heterocysts (>20% of total cells), which were barely functional probably due to a weak expression of nitrogenase. In addition, heterocysts of the mutant strain did not display the usual cellular structure of nitrogen-fixative cells. This unveiled why the mutant strain was not able to grow under nitrogen starvation.

A novel Ca2+-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120.

Ca2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca2+-binding protein which was called the Ca2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca2+, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes.

Glycolipid composition of the heterocyst envelope of Anabaena sp. PCC 7120 is crucial for diazotrophic growth and relies on the UDP-galactose 4-epimerase HgdA.

The nitrogenase complex in the heterocysts of the filamentous freshwater cyanobacterium Anabaenasp. PCC 7120 fixes atmospheric nitrogen to allow diazotrophic growth. The heterocyst cell envelope protects the nitrogenase from oxygen and consists of a polysaccharide and a glycolipid layer that are formed by a complex process involving the recruitment of different proteins. Here, we studied the function of the putative nucleoside-diphosphate-sugar epimerase HgdA, which along with HgdB and HgdC is essential for deposition of the glycolipid layer and growth without a combined nitrogen source. Using site-directed mutagenesis and single homologous recombination approach, we performed a thoroughly functional characterization of HgdA and confirmed that the glycolipid layer of the hgdAmutant heterocyst is aberrant as shown by transmission electron microscopy and chemical analysis. The hgdA gene was expressed during late stages of the heterocyst differentiation. GFP-tagged HgdA protein localized inside the heterocysts. The purified HgdA protein had UDP-galactose 4-epimerase activity in vitro. This enzyme could be responsible for synthesis of heterocyst-specific glycolipid precursors, which could be transported over the cell wall by the ABC transporter components HgdB/HgdC.

Cyanobacterial antimetabolite 7-deoxy-sedoheptulose blocks the shikimate pathway to inhibit the growth of prototrophic organisms.

Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-D-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-D-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent.

The Individual and Combined Effects of the Cyanotoxins, Anatoxin-a and Microcystin-LR, on the Growth, Toxin Production, and Nitrogen Fixation of Prokaryotic and Eukaryotic Algae.

Globally, eutrophication and warming of aquatic ecosystems has increased the frequency and intensity of cyanobacterial blooms and their associated toxins, with the simultaneous detection of multiple cyanotoxins often occurring. Despite the co-occurrence of cyanotoxins such as microcystins and anatoxin-a (ATX) in water bodies, their effects on phytoplankton communities are poorly understood. The individual and combined effects of microcystin-LR (MC-LR) and ATX on the cyanobacteria Microcystis spp., and Anabaena variabilis (a.k.a. Trichormus variabilis), and the chlorophyte, Selenastrum capricornutum were investigated in the present study. Cell density, chlorophyll-a content, and the maximum quantum efficiency of photosystem II (Fv/Fm) of Microcystis cells were generally lowered after exposure to ATX or MC-LR, while the combined treatment with MC-LR and ATX synergistically reduced the chlorophyll-a concentration of Microcystis strain LE-3. Intracellular levels of microcystin in Microcystis LE-3 significantly increased following exposure to MC-LR + ATX. The maximum quantum efficiency of photosystem II of Anabaena strain UTEX B377 declined during exposure to the cyanotoxins. Nitrogen fixation by Anabaena UTEX B377 was significantly inhibited by exposure to ATX, but was unaffected by MC-LR. In contrast, the combination of both cyanotoxins (MC-LR + ATX) caused a synergistic increase in the growth of S. capricornutum. While the toxins caused an increase in the activity of enzymes that scavenge reactive oxygen species in cyanobacteria, enzyme activity was unchanged or decreased in S. capricornutum. Collectively this study demonstrates that MC-LR and ATX can selectively promote and inhibit the growth and performance of green algae and cyanobacteria, respectively, and that the combined effect of these cyanotoxins was often more intense than their individual effects on some strains. This suggests that the release of multiple cyanotoxins in aquatic ecosystems, following the collapse of blooms, may influence the succession of plankton communities.

Isolation and characterization of high temperature tolerant mutant from the cyanobacterium Anabaena doliolum.

The mutant strain of the diazotrophic cyanobacterium Anabaena doliolum able to tolerate high temperature was isolated by induced mutation techniques using ethyl methane sulphonate. This mutant strain exhibited higher temperature tolerance than the wild type. The wild type was able tolerate temperature up to 40 °C whereas the mutant was able to grow at an elevated temperature of 48 °C. This mutant exhibited higher growth rate, heterocyst frequency, and nitrogen fixation. Mutant strains exhibited comparable levels of chlorophyll, phycocyanin, PS II activity, and O2 evolution as compared to unexposed control. Results also showed that the mutant accumulated low levels of peroxides and lipid peroxidation products with enhanced activity of antioxidant enzymes. The FAME analysis revealed quantitative and qualitative changes in the profile of fatty acids in the mutant strain. Maximum number of saturated fatty acids was observed in the mutant strain followed by control whereas the wild type exposed to elevated temperature showed least diversity of fatty acids. Enhanced level of antioxidant enzymes coupled with efficient modulation of fatty acid profile could therefore enhance the mutant to resist the high temperature stress. The results could be exploited further to decipher molecular mechanisms underlying the temperature tolerance and enhancing the utility of A. doliolum as efficient biofertilizer for rice paddy keeping in view of the future climatic change scenario.