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9 páginas.- 6 figuras.- 2 tabla.- 1 referencia.- Supplementary data to this article can be found online at https://doi. org/10.1016/j.jclepro.2023.136356, Coagulation-flocculation is a critical treatment to reduce operational problems at Drinking Water Treatment Plants (DWTPs), created by episodes of cyanobacterial blooms in surface waters. There is a need for the search of good coaguflocculants that avoid release of intracellular toxins for a safer sustainable production. Clay-polymer interactions were examined for the development of efficient composites for their use as coaguflocculants for Microcystis aeruginosa (M. aeruginosa) suspensions. Polymers derived from poly-4-vinylpyridine (PVP) were quaternized on the pyridinic N by introducing methyl and hydroxyethyl moieties. These polymers were toxic per se; however, composites prepared either from their sorption or grafting on the clay surface of a montmorillonite exhibited null toxicity, The combination of infrared and X-ray diffraction data with thermal analysis showed a train conformation of the sorbed polymers on the clay surface whereas a brush conformation was elucidated for the grafted polymers. Only the composite prepared from grafting and subsequent quaternization with methyls was an efficient coaguflocculant, due to its high positive surface potential (+39 mV) which allowed a close contact between the quaternary moieties of the brushes and the negative cell wall. A patch flocculation mech-anism was involved, with the riks of resuspension of the coagulated cells if the composite added in excess. The optimum ratio between the amount of coagulated cyanobacteria and this composite expressed as equivalent to polymer content was 7.2 x 107 cells/mg polymer. This ratio was determined in axenic cultures of M. aeruginosa, but was reduced 5-fold in natural surface waters due to natural organic matter (NOM) content and heterogeneity. This study has demonstrated the relevance of the type of modification of the clay mineral surface with polymers to obtain good coaguflocculants of cyanobacterial cells, that can be extended to other microorganisms., This work has received financial support from the Spanish Ministry of Science and Innovation (project CTM2016-77168-R). The authors also acknowledge the Services of the University of Seville (CITIUS) of Biology for flow cytometer analysis, and of Microanalysis and Functional Characterisation for determination of zeta potentials, loading of the polymers and MC-LR analysis. They also acknowledge the Micro-scopy Service of the CIC Isla de la Cartuja.

Biological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein AtpQ, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of AtpQ becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of AtpQto the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far western blots identified subunits of ATP synthase as interacting partners of AtpQ. ATP hydrolysis assays with isolated membrane fractions, as well as purified ATP synthase complexes, demonstrated that AtpQ inhibits ATPase activity in a dose-dependent manner similar to the F0F1-ATP synthase inhibitor N,N-dicyclohexylcarbodimide. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration and indicate that ATP synthase activity can be controlled in surprisingly different ways.

SummaryBiological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein AtpΘ, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of AtpΘ becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of AtpΘ to the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far Western blots identified subunits of ATP synthase as interacting partners of AtpΘ. ATP hydrolysis assays with isolated membrane fractions as well as purified ATP synthase complexes demonstrated that AtpΘ inhibits ATPase activity in a dose-dependent manner similar to the F0F1-ATP synthase inhibitor N,N-dicyclohexylcarbodimide. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration and indicate that ATP synthase activity can be controlled in surprisingly different ways.

Biological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein AtpΘ, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of AtpΘ becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of AtpΘ to the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far western blots identified subunits of ATP synthase as interacting partners of AtpΘ. ATP hydrolysis assays with isolated membrane fractions, as well as purified ATP synthase complexes, demonstrated that AtpΘ inhibits ATPase activity in a dose-dependent manner similar to the F

10 páginas, 4 figuras., Cyanobacteria are phototrophic bacteria carrying out oxygen-producing photosynthesis. Indeed, cyanobacteria were the inventors of oxygenic photosynthesis carried out by eukaryotic algae and plants. Besides showing the capability of building their cellular carbon from carbon dioxide, available in the atmosphere, several strains of cyanobacteria have also acquired the ability to fix molecular dinitrogen (N2). As the enzyme responsible for nitrogen fixation (nitrogenase) is highly sensitive towards oxygen, nitrogen fixation and oxygenic photosynthesis cannot take place simultaneously in cyanobacterial cells. To solve this problem, some filamentous strains are able to restrict N2 fixation to a special cell type, the heterocyst. Heterocysts are specialised, morphologically distinct, terminally differentiated cells that develop, in the absence of alternative sources of combined nitrogen, mostly in a semiregular pattern along the filament. Thus, a filament containing heterocysts provides division of labour between photosynthetic carbon dioxide fixation (in vegetative cells) and anaerobic N2 fixation (in heterocysts). These cyanobacteria represent true multicellular organisms with profound morphological cell differentiation and sophisticated intercellular communication systems., The support to I.M. by Deutsche Forschungsgemeinschaft at the University of Regensburg and Tübingen is gratefully acknowledged.

In the absence of fixed nitrogen, some filamentous cyanobacteria differentiate heterocysts, specialized cells devoted to fixing atmospheric nitrogen (N2). This differentiation process is controlled by the global nitrogen regulator NtcA and involves extensive metabolic reprogramming, including shutdown of photosynthetic CO2 fixation in heterocysts, to provide a microaerobic environment suitable for N2 fixation. Small regulatory RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. In cyanobacteria, responding to nitrogen deficiency involves transcribing several nitrogen-regulated sRNAs. Here, we describe the participation of nitrogen stress-inducible RNA 4 (NsiR4) in post-transcriptionally regulating the expression of two genes involved in CO2 fixation via the Calvin cycle: glpX, which encodes bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). Using a heterologous reporter assay in Escherichia coli, we show that NsiR4 interacts with the 5′-untranslated region (5′-UTR) of glpX and pgk mRNAs. Overexpressing NsiR4 in Nostoc sp. PCC 7120 resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs, further supporting that NsiR4 negatively regulates these two enzymes. In addition, using a gfp fusion to the nsiR4 promoter, we show stronger expression of NsiR4 in heterocysts than in vegetative cells, which could contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, a nitrogen-regulated sRNA, represents an additional link between nitrogen control and CO2 assimilation., The small RNA nitrogen stress-inducible RNA 4 (NsiR4) regulates two enzymes involved in CO2 fixation in cyanobacteria.

Cyanobacteria are photosynthetic bacteria that populate widely different habitats. Accordingly, cyanobacteria exhibit a wide spectrum of lifestyles, physiologies, and morphologies and possess genome sizes and gene numbers which may vary by up to a factor of ten within the phylum. Consequently, large differences exist between individual species in the size and complexity of their regulatory networks. Several non-coding RNAs have been identified that play crucial roles in the acclimation responses of cyanobacteria to changes in the environment. Some of these regulatory RNAs are conserved throughout the cyanobacterial phylum, while others exist only in a few taxa. Here we give an overview on characterized regulatory RNAs in cyanobacteria, with a focus on regulators of photosynthesis, carbon and nitrogen metabolism. However, chances are high that these regulators represent just the tip of the iceberg.

Upon nitrogen starvation, filamentous cyanobacteria develop heterocysts, specialized cells devoted to the fixation of atmospheric nitrogen. Differentiation of heterocyst at semi-regular intervals along the filaments requires complex structural and functional changes that are under the control of the master transcriptional regulator HetR. NsiR1 (nitrogen stress-induced RNA 1) is a HetR-dependent non-coding RNA that is expressed specifically in heterocysts from a very early stage of differentiation. In the genome of Nostoc sp. PCC 7120 there are 12 tandem copies of nsiR1 (nsiR1.1 to nsiR1.12), seven of them with identical sequence (nsiR1.3 to nsiR1.9) and the others slightly divergent. nsiR1.1 is transcribed antisense to the 5′ UTR of hetF, a gene required for heterocyst development. Here, we show that binding of NsiR1.1 inhibits translation of the hetF mRNA by inducing structural changes in its 5′ UTR. Altered levels of NsiR1 result in different phenotypic alterations including enlarged cell size and delayed heterocyst development that could be related to a reduced amount of HetF., This work was supported by grants BFU2013‐48282‐C2‐1‐P from Ministerio de Economía y Competitividad, and BFU2016‐74943‐C2‐1‐P from Agencia Estatal de Investigación (AEI), Ministerio de Economía, Industria y Competitividad, both cofinanced by the European Regional Development Fund, to A.M.M‐P. M.B.‐A. is the recipient of a predoctoral contract from Ministerio de Educación, Cultura y Deporte, Spain (FPU014/05123 and EST16‐00088)

Cyanobacteria, the most ancient and abundant photoautotrophs on Earth, produce the greenhouse gas methane during photosynthesis., Evidence is accumulating to challenge the paradigm that biogenic methanogenesis, considered a strictly anaerobic process, is exclusive to archaea. We demonstrate that cyanobacteria living in marine, freshwater, and terrestrial environments produce methane at substantial rates under light, dark, oxic, and anoxic conditions, linking methane production with light-driven primary productivity in a globally relevant and ancient group of photoautotrophs. Methane production, attributed to cyanobacteria using stable isotope labeling techniques, was enhanced during oxygenic photosynthesis. We suggest that the formation of methane by cyanobacteria contributes to methane accumulation in oxygen-saturated marine and limnic surface waters. In these environments, frequent cyanobacterial blooms are predicted to further increase because of global warming potentially having a direct positive feedback on climate change. We conclude that this newly identified source contributes to the current natural methane budget and most likely has been producing methane since cyanobacteria first evolved on Earth.

Yfr1 is a strictly conserved small RNA in cyanobacteria. A bioinformatic prediction to identify possible interactions of Yfr1 with mRNAs was carried out by using the sequences of Yfr1 from several heterocyst-forming strains, including Nostoc sp. strain PCC 7120. The results of the prediction were enriched in genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover. Heterologous expression assays with Escherichia coli demonstrated direct interactions of Yfr1 with mRNAs of 11 of the candidate genes. The expression of 10 of them (alr2458, alr4550, murC, all4829, all2158, mraY, alr2269, alr0834, conR, patN) was repressed by interaction with Yfr1, whereas the expression of amiC2, encoding an amidase, was increased. The interactions between Yfr1 and the 11 mRNAs were confirmed by site-directed mutagenesis of Yfr1. Furthermore, a Nostoc strain with reduced levels of Yfr1 had larger amounts of mraY and murC mRNAs, supporting a role for Yfr1 in the regulation of those genes. Nostoc strains with either reduced or increased expression of Yfr1 showed anomalies in cell wall completion and were more sensitive to vancomycin than the wild-type strain. Furthermore, growth in the absence of combined nitrogen, which involves the differentiation of heterocysts, was compromised in the strain overexpressing Yfr1, and filaments were broken at the connections between vegetative cells and heterocysts. These results indicate that Yfr1 is an important regulator of cell wall homeostasis and correct cell wall remodeling during heterocyst differentiation

Recently, we studied glucose-nucleobase pairs, a binding motif found in aminoglycoside–RNA recognition. DNA duplexes with glucose as a nucleobase were able to hybridize and were selective for purines. They were less stable than natural DNA but still fit well on regular B-DNA. These results opened up the possible use of glucose as a non-aromatic DNA base mimic. Here, we have studied the incorporation and thermal stability of glucose with different types of anchoring units and alternative apolar sugar-nucleobase pairs. When we explored butanetriol instead of glycerol as a wider anchoring unit, we did not gain duplex thermal stability. This result confirmed the necessity of a more conformationally restricted linker to increase the overall duplex stability. Permethylated glucose-nucleobase pairs showed similar stability to glucoside-nucleobase pairs but no selectivity for a specific nucleobase, possibly due to the absence of hydrogen bonds between them. The three-dimensional structure of the duplex solved by NMR located both, the hydrophobic permethylated glucose and the nucleobase, inside the DNA helix as in the case of glucose-nucleobase pairs. Quantum chemical calculations on glucose-nucleobase pairs indicate that the attachment of the sugar to the DNA skeleton through the OH1 or OH4 positions yields the highest binding energies. Moreover, glucose was very selective for guanine when attached through OH1 or OH4 to the DNA. Finally, we examined DNA polymerase insertion of nucleotides in front of the saccharide unit. KF− polymerase from E. coli inserted A and G opposite glc and 6dglc with low efficiency but notable selectivity. It is even capable of extending the new pair although its efficiency depended on the DNA sequence. In contrast, Bst 2.0, SIII and BIOTAQ™ DNA polymerases seem to display a loop-out mechanism possibly due to the flexible glycerol linker used instead of deoxyribose., We thank the Ministerio de Economia y Competitividad (CTQ2011-15203-E, CTQ2012-35360, CTQ2014-52588-R, CTQ2015- 64275-P, BFU2014-52864-R, and BFU2017-89707-P) and the Netherlands Organization for Scientific Research (NWO-CW and NWO-EW) for financial support. E. V. C. thanks the Ministerio de Educación, Cultura y Deporte for an FPU fellowship and Cost Action CM1005 for an STSM grant. R. L. is a recipient of a Talent Hub fellowship from Junta de Andalucia

Summary Heterocystous cyanobacteria such as Nostoc sp. are filamentous photosynthetic organisms that, in response to nitrogen deficiency, undergo a differentiation process transforming certain, semi-regularly spaced cells into heterocysts, devoted to nitrogen fixation. During transition to a nitrogen-fixing regime, growth of most vegetative cells in the filament is temporarily arrested due to nutritional deprivation, but developing heterocysts require intense transcriptional activity. Therefore, the coexistence of arrested vegetative cells and actively developing prospective heterocysts relies on the simultaneous operation of somewhat opposite transcriptional programs. We have identified genes with multiple nitrogen-responsive transcriptional starts appearing in seemingly paradoxical combinations. For instance, sigA, encoding the RNA polymerase housekeeping sigma factor, is transcribed from one major nitrogen stress-repressed promoter and from a second, nitrogen stress-induced promoter. Here we show that both promoters are expressed with complementary temporal dynamics. Using a gfp reporter we also show that transcription from the inducible promoter takes place exclusively in differentiating heterocysts and is already detected before any morphological or fluorescence signature of differentiation is observed. Tandem promoters with opposite dynamics could operate a compensatory mechanism in which repression of transcription from the major promoter operative in vegetative cells is offset by transcription from a new promoter only in developing heterocyst. This article is protected by copyright. All rights reserved.

Upon nitrogen deficiency, some filamentous cyanobacteria differentiate specialized cells, called heterocysts, devoted to N2 fixation. Heterocysts appear regularly spaced along the filaments and exhibit structural and metabolic adaptations, such as loss of photosynthetic CO2 fixation or increased respiration, to provide a proper microaerobic environment for its specialized function. Heterocyst development is under transcriptional control of the global nitrogen regulator NtcA and the specific regulator HetR. Transcription of a large number of genes is induced or repressed upon nitrogen deficiency specifically in cells undergoing differentiation. In recent years, the HetR regulon has been described to include heterocyst-specific trans-acting small RNAs and antisense RNAs (asRNAs), suggesting that there is an additional layer of post-transcriptional regulation involved in heterocyst development. Here, we characterize in the cyanobacterium Nostoc (Anabaena) sp. PCC 7120 an asRNA, that we call as_glpX, transcribed within the glpX gene encoding the Calvin cycle bifunctional enzyme sedoheptulose-1,7- bisphosphatase/fructose-1,6-bisphosphatase (SBPase). Transcription of as_glpX is restricted to heterocysts and is induced very early during the process of differentiation. Expression of as_glpX RNA promotes the cleavage of the glpX mRNA by RNase III, resulting in a reduced amount of SBPase. Therefore, the early expression of this asRNA could contribute to the quick shut-down of CO2 fixation in those cells in the filament that are undergoing differentiation into heterocysts. In summary, as_glpX is the first naturally occurring asRNA shown to rapidly and dynamically regulate metabolic transformation in Nostoc heterocysts. The use of antisense transcripts to manipulate gene expression specifically in heterocysts could became a useful tool for metabolic engineering in cyanobacteria.

Nitrogen is frequently limiting microbial growth in the environment. As a response, many filamentous cyanobacteria differentiate heterocysts, cells devoted to N fixation. Heterocyst differentiation is under the control of the master regulator HetR. Through the characterization of the HetR-dependent transcriptome in Nostoc sp. PCC 7120, we identified the new candidate genes likely involved in heterocyst differentiation. According to their maximum induction, we defined E-DIF (early in differentiation) and L-DIF (late in differentiation) genes. Most of the genes known to be involved in the critical aspects of heterocyst differentiation or function were also classified into these groups, showing the validity of the approach. Using fusions to gfp, we verified the heterocyst-specific transcription of several of the found genes, antisense transcripts and potentially trans-acting sRNAs. Through comparative sequence analysis of promoter regions, we noticed the prevalence of the previously described DIF1 motif and identified a second motif, called DIF2, in other promoters of the E-DIF cluster. Both motifs are widely conserved in heterocystous cyanobacteria. We assigned alr2522 as a third member, besides nifB and nifP, to the CnfR regulon. The elements identified here are of interest for understanding cell differentiation, engineering of biological nitrogen fixation or production of O-sensitive molecules in cyanobacteria.

Small regulatory RNAs (sRNAs) are currently considered as major post-transcriptional regulators of gene expression in bacteria. The interplay between sRNAs and transcription factors leads to complex regulatory networks in which both transcription factors and sRNAs may appear as nodes. In cyanobacteria, the responses to nitrogen availability are controlled at the transcriptional level by NtcA, a CRP/FNR family regulator. In this study, we describe an NtcA-regulated sRNA in the cyanobacterium Nostoc sp. PCC 7120, that we have named NsrR1 (nitrogen stress repressed RNA1). We show sequence specific binding of NtcA to the promoter of NsrR1. Prediction of possible mRNA targets regulated by NsrR1 allowed the identification of nblA, encoding a protein adaptor for phycobilisome degradation under several stress conditions, including nitrogen deficiency. We demonstrate specific interaction between NsrR1 and the 5′-UTR of the nblA mRNA, that leads to decreased expression of nblA. Because both NsrR1 and NblA are under transcriptional control of NtcA, this regulatory circuit constitutes a coherent feed-forward loop, involving a transcription factor and an sRNA.

Evidence is accumulating to challenge the paradigm that biogenic methanogenesis, traditionally considered a strictly anerobic process, is exclusive to Archaea. Here we demonstrate that Cyanobacteria living in marine, freshwater and terrestrial environments produce methane at substantial rates under light and dark oxic and anoxic conditions, forming a link between light driven primary productivity and methane production in globally relevant group of phototrophs. Biogenic methane production was enhanced during oxygenic photosynthesis and directly attributed to the cyanobacteria by applying stable isotope labelling techniques. We suggest that formation of methane by Cyanobacteria may contribute to methane accumulation in oxygen-saturated surface waters of marine and freshwater ecosystems. Moreover, in these environments, cyanobacterial blooms already do, and might further occur more frequently during future global warming and thus have a direct feedback on climate change. We further highlight that cyanobacterial methane production not only affects recent and future global methane budgets, but also has implications for inferences on Earth’s methane budget for the last 3.5 billion years, when this phylum is thought to have first evolved.

Novel CRISPR-Cas systems possess substantial potential for genome editing and manipulation of gene expression. The types and numbers of CRISPR-Cas systems vary substantially between different organisms. Some filamentous cyanobacteria harbor > 40 different putative CRISPR repeat-spacer cassettes, while the number of cas gene instances is much lower. Here we addressed the types and diversity of CRISPR-Cas systems and of CRISPR-like repeat-spacer arrays in 171 publicly available genomes of multicellular cyanobacteria. The number of 1328 repeat-spacer arrays exceeded the total of 391 encoded Cas1 proteins suggesting a tendency for fragmentation or the involvement of alternative adaptation factors. The model cyanobacterium Anabaena sp. PCC 7120 contains only three cas1 genes but hosts three Class 1, possibly one Class 2 and five orphan repeat-spacer arrays, all of which exhibit crRNA-typical expression patterns suggesting active transcription, maturation and incorporation into CRISPR complexes. The CRISPR-Cas system within the element interrupting the Anabaena sp. PCC 7120 fdxN gene, as well as analogous arrangements in other strains, occupy the genetic elements that become excised during the differentiation-related programmed site-specific recombination. This fact indicates the propensity of these elements for the integration of CRISPR-cas systems and points to a previously not recognized connection. The gene all3613 resembling a possible Class 2 effector protein is linked to a short repeat-spacer array and a single tRNA gene, similar to its homologs in other cyanobacteria. The diversity and presence of numerous CRISPR-Cas systems in DNA elements that are programmed for homologous recombination make filamentous cyanobacteria a prolific resource for their study.

9 páginas.- 7 figuras.- 4 tablas.- 42 referencias.- Supplementary data to this article can be found online at https://doi.org/10.1016/j.jclepro.2019.117964, Cyanobacterial blooming episodes in surface waters used for drinking purposes are increasing due to eutrophication of water ecosystems. Water treatments should be optimised to remove phytoplankton cells as well as their associated toxins. The performance of a multicell electrocoagulation reactor operating under a continuous flow and coupled with flocculation for cyanobacteria removal was examined. Electrocoagulation of a suspension of the unicellular cyanobacteria Microcystis aeruginosa and its related toxin microcystin-LR, as a model of toxic cyanobacterium and cyanotoxin, respectively, showed that cell removal occurred through charge neutralisation with the Al hydroxides generated in the system, whereas the toxin did not undergo electrolytical processes. Cell inactivation was a function of the charge loading and the cell population. Inactivation of a 105 cells/mL suspension was 34% for a charge loading of 50 C/L whereas only 3% was achieved by increasing cyanobacteria concentration by one-order of magnitude. Flocculation of the electrocoagulated suspensions with anionic polyelectrolytes revealed a bridging mechanism, whereas an electrostatic patch aggregation was involved with cationic flocculants. Electrocoagulation/flocculation of a cyanobacterial blooming surface water (about 106 cells/mL) showed quite good concordance of cell inactivation values (9.7–10.7%) with those detected previously with Microcystis aeruginosa (5.4%). However, the performance of the flocculants was slightly worse as determined from the residual turbidity values (17% vs. 7% in pure cultures of M. aeruginosa) due to the different compositions of the extracellular organic matter. © 2019 Elsevier Ltd, Financial support was received by the grant CTM2016-77168-R from the Spanish Ministry of Science, Innovation and Universities supported by the European Regional Development Fund (FEDER) . The authors also acknowledge the Services of the University of Seville (CITIUS) of Biology for flow cytometer analysis, and Microanalysis and Functional Characterisation Services for determination of zeta potentials and floc sizes. The authors also thank Dr. González-Grau for his assistance in the analysis of cyanobacteria genera. Cyclus ID (Moron de la Frontera, Spain) is acknowledged by its assistance in the construction of the EC equipment. Appendix A

Small RNAs (sRNAs) are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behavior and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq) facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells). Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8), in developing heterocysts is also demonstrated.

There are several instances of cellular differentiation in prokaryotes, including the formation of spores in Bacillus, the fruiting bodies of Myxococcus, and the stalked cells of Caulobacter. The vegetative cells of particular filamentous cyanobacteria can differentiate into three different cell types: N(2)-fixing heterocysts, spore-like akinetes, and motile hormogonia. Heterocysts are crucial for the ability of these photosynthetic bacteria to fix N(2) because they keep the oxygen-labile nitrogenase away from the photosynthetically produced O(2). Heterocysts are morphologically and functionally distinct from vegetative cells in the filament. Their differentiation relies on sophisticated intercellular communication and is tightly regulated. Analyzed by classical mutagenesis for decades, heterocyst differentiation is now being approached by large-scale methodologies, leading to the identification of new elements that might be important in the process.

Bacteria secrete small signal molecules into the environment that induce self and neighbour gene expression. This phenomenon, termed quorum sensing, allows cooperative behaviours that increase the fitness of the group. The best-studied signal molecules are the N-acylhomoserine lactones (AHLs), secreted by a growing number of bacterial species including important pathogen species such as Pseudomonas aeruginosa. These molecules have recently been proposed to have properties other than those of signalling, functioning as iron quelants or antibiotics. As the presence of an acylase capable of inactivating long-chain AHLs in Anabaena sp. PCC7120 could constitute a defence mechanism against these molecules, in this work we analyse the effects of different AHLs varying in length and substitutions on the growth and nitrogen metabolism of the cyanobacterium Anabaena sp. PCC7120. All the AHLs tested strongly inhibited nitrogen fixation. The inhibition seems to take place at post-transcriptional level, as no effect on heterocyst differentiation or on the expression of nitrogenase was observed. Moreover, N-(3-oxodecanoyl)-l-homoserine lactone (OC10-HSL) showed a specific cytotoxic effect on this cyanobacterium in the presence of a combined nitrogen source, but the mechanism involved seems to be different from that described so far for tetramic acid derivatives of oxo-substituted AHLs. These results suggest a variety of new unexpected activities for AHLs, at least on cyanobacterial populations.

Heterocyst-forming cyanobacteria are able to perform oxygenic photosynthesis and nitrogen fixation simultaneously in the same filament, by restricting the highly O2-sensitive nitrogenase to specialized cells, the heterocysts. A remarkable change in morphology and metabolism accompanies the differentiation of heterocysts, which only occurs when no source of combined nitrogen is available. In this study, we characterized DevT (Alr4674), a putative protein phosphatase from Anabaena PCC 7120. Mutants defective in devT are able to form morphologically mature heterocysts, which however cannot fix N2, and the mutant cannot survive without a source of combined nitrogen. DevT shows homology to phosphatases of the PPP family and displays a Mn2+-dependent phosphatase activity that can be inhibited by phosphatase inhibitors and oxidizing conditions. DevT is constitutively expressed in both vegetative cells and heterocysts, and is not regulated by NtcA. The heterocyst regulator HetR may exert a certain inhibition on the expression of devT. Under diazotrophic growth conditions, DevT protein accumulates specifically in mature heterocysts. Therefore DevT plays a still unknown role in a late essential step of heterocyst differentiation. © 2010 SGM.

Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5′UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus NH+4 assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient

In cyanobacteria, DNA-binding proteins from starved cells (Dps) play an important role in the cellular response to oxidative and nutritional stresses. In this study, we have characterized the cell-type specificity and the promoter regions of two orthologous dps genes, Npun_R5799 in Nostoc punctiforme and alr3808 in Anabaena sp. PCC 7120. A transcriptional start site (TSS), identical in location to the previously identified proximal TSS of alr3808, was identified for Npun_R5799 under both combined nitrogen supplemented and N2-fixing growth conditions. However, only alr3808 was also transcribed from a second distal TSS. Sequence homologies suggest that the promoter region containing the distal TSS is not conserved upstream of orthologous genes among heterocyst-forming cyanobacteria. The analysis of promoter GFP-reporter strains showed a different role in governing cell-type specificity between the proximal and distal promoter of alr3808. We here confirmed the heterocyst specificity of the distal promoter of alr3808 and described a very early induction of its expression during proheterocyst differentiation. In contrast, the complete promoters of both genes were active in all cells. Even though Npun_R5799 and alr3808 are orthologs, the regulation of their respective expression differs, indicating distinctions in the function of these cyanobacterial Dps proteins depending on the strain and cell type.

Expression of the glnA gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N 2 -fixing cyanobacterium Anabaena sp. strain PCC 7120, glnA is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNA I originates from a canonical NtcA-dependent promoter (P 1 ) and RNA II originates from a σ 70 -type promoter (P 2 ), RNA IV is influenced by NtcA but the corresponding promoter (P 3 ) does not have the structure of NtcA-activated promoters. Using RNA isolated from Anabaena filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNA V , which has previously been detected only in in vitro transcription assays and should originate from P 4 . However, in heterocysts, which are differentiated cells specialized in N 2 fixation, RNA I was the almost exclusive glnA transcript. Analysis of P glnA :: lacZ fusions containing different fragments of the glnA upstream region confirmed that fragments carrying P 1 , P 2 , or P 3 and P 4 have the ability to promote transcription. Mutation of the NtcA-binding site in P 1 eliminated P 1 -directed transcription and allowed increased use of P 2 . The NtcA-binding site in the P 1 promoter and binding of NtcA to this site appear to be key factors in determining glnA gene expression in vegetative cells and heterocysts.

Some filamentous cyanobacteria can undergo a variety of cellular differentiation processes that permit their better adaptation to certain environmental conditions. These processes include the differentiation of hormogonia, short filaments aimed at the dispersal of the organism in the environment, of akinetes, cells resistant to various stress conditions, and of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. NtcA is a transcriptional regulator that operates global nitrogen control in cyanobacteria by activating (and in some cases repressing) many genes involved in nitrogen assimilation. NtcA is required for the triggering of heterocyst differentiation and for subsequent steps of its development and function. This requirement is based on the role of NtcA as an activator of the expression of hetR and other multiple genes at specific steps of the differentiation process. The products of these genes effect development as well as the distinct metabolism of the mature heterocyst. The different features found in the NtcA-dependent promoters, together with the cellular level of active NtcA protein, should have a role in the determination of the hierarchy of gene activation during the process of heterocyst differentiation.

Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step-down depends on NtcA. The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions -728 and -271) was found to be dependent on NtcA, and the use of the tsp located at position -271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen-fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions -180 and -49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step-down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA-dependent tsp was, however, not dependent on HetR.

Three genes, coxB, coxA , and coxC , found in a clone from a gene library of the cyanobacterium Anabaena variabilis strain ATCC 29413, were identified by hybridization with an oligonucleotide specific for aa 3 -type cytochrome c oxidases. Deletion of these genes from the genome of A. variabilis strain ATCC 29413 FD yielded strain CSW1, which displayed no chemoheterotrophic growth and an impaired cytochrome c oxidase activity. Photoautotrophic growth of CSW1, however, was unchanged, even with dinitrogen as the nitrogen source. A higher cytochrome c oxidase activity was detected in membrane preparations from dinitrogen-grown CSW1 than from nitrate-grown CSW1, but comparable activities of respiratory oxygen uptake were found in the wild type and in CSW1. Our data indicate that the identified cox gene cluster is essential for fructose-dependent growth in the dark, but not for growth on dinitrogen, and that other terminal respiratory oxidases are expressed in this cyanobacterium. Transcription analysis showed that coxBAC constitutes an operon which is expressed from two transcriptional start points. The use of one of them was stimulated by fructose.

The devBCA operon, encoding subunits of an ATP-binding cassette exporter, is essential for differentiation of N 2 -fixing heterocysts in Anabaena spp. Nitrogen deficiency-dependent transcription of the operon and the use of its transcriptional start point, located 762 ( Anabaena variabilis strain ATCC 29413-FD) or 704 ( Anabaena sp. strain PCC 7120) bp upstream of the translation start site, were found to require the global nitrogen transcriptional regulator NtcA. Furthermore, NtcA was shown to bind in vitro to the promoter of devBCA .

Nitrogen is a quantitatively important bioelement which is incorporated into the biosphere through assimilatory processes carried out by microorganisms and plants. Numerous nitrogencontaining compounds can be used by different organisms as sources of nitrogen. These include, for instance, inorganic ions like nitrate or ammonium and simple organic compounds like urea, amino acids, and some nitrogen-containing bases. Additionally, many bacteria are capable of fixing N 2. Nitrogen control is a phenomenon that occurs widely among microorganisms and consists of repression of the pathways of assimilation of some nitrogen sources when some other, more easily assimilated source of nitrogen is available to the cells. Ammonium is the preferred nitrogen source for most bacteria, but glutamine is also a very good source of nitrogen for many microorganisms. Two thoroughly investigated nitrogen control systems are the NtrB-NtrC two-component regulatory system found in enterics and some other proteobacteria (80) and the GATA family global nitrogen control transcription factors of yeast and some fungi (75). Novel nitrogen control systems have, however, been identified in bacteria other than the proteobacteria, like Bacillus subtilis (26), Corynebacterium glutamicum (52), and the cyanobacteria. The cyanobacterial system is the subject of this review. The cyanobacteria are prokaryotes that belong to the Bacteria domain and are characterized by the ability to perform oxygenic photosynthesis. Cyanobacteria have a wide ecological distribution, and they occupy a range of habitats, which includes vast oceanic areas, temperate soils, and freshwater lakes, and even extreme habitats like arid deserts, frigid lakes, or hot springs. Photoautotrophy, fixing CO 2 through the Calvin cycle, is the dominant mode of growth of these organisms (109). A salient feature of the intermediary metabolism of cyanobacteria is their lack of 2-oxoglutarate dehydrogenase (109). As a consequence, they use 2-oxoglutarate mainly as a substrate for the incorporation of nitrogen, a metabolic arrangement that may have regulatory consequences. Notwithstanding their rather homogeneous metabolism, cyanobacteria exhibit remarkable morphological diversity, being found as either unicellular or filamentous forms and exhibiting a number of cell differentiation processes, some of which take place in response to defined environmental cues, as is the case for the differentiation of N 2-fixing heterocysts (109). Nitrogen control in cyanobacteria is mediated by NtcA, a transcriptional regulator which belongs to the CAP (the catabolite gene activator or cyclic AMP [cAMP] receptor protein) family and is therefore different from the well-characterized Ntr system. Interestingly, however, the signal transduction P II protein, which plays a key role in Ntr regulation, is found in cyanobacteria but with characteristics which differentiate it from proteobacterial P II. In the following paragraphs, we shall first briefly summarize our current knowledge of the cyanobacterial nitrogen assimilation pathways and of what is known about their regulation at the protein level. This description will introduce most of the known cyanobacterial nitrogen assimilation genes. We shall then describe the ntcA gene and the NtcA protein themselves to finally discuss NtcA function through a survey of the NtcA-regulated genes which participate in simple nitrogen assimilation pathways or in heterocyst differentiation and function.

The heterocyst is the site of nitrogen fixation in aerobically grown cultures of some filamentous cyanobacteria. Heterocyst development in Anabaena sp. strain PCC 7120 is dependent on the global nitrogen regulator NtcA and requires, among others, the products of the hetR and hetC genes. Expression of hetC , tested by RNA- DNA hybridization, was impaired in an ntcA mutant. A nitrogen-regulated, NtcA-dependent putative transcription start point was localized at nucleotide −571 with respect to the hetC translational start. Sequences upstream from this transcription start point exhibit the structure of the canonical cyanobacterial promoter activated by NtcA, and purified NtcA protein specifically bound to a DNA fragment containing this promoter. Activation of expression of hetC during heterocyst development appears thus to be directly operated by NtcA. NtcA-mediated activation of hetR expression was not impaired in a hetC mutant, indicating that HetC is not an NtcA-dependent element required for hetR induction.

Determination of the putative transcription start points of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst-forming cyanobacteria Anabaena sp. PCC 7119 and PCC 7120 showed that this gene is transcribed from two promoters, one constitutively used under different conditions of nitrogen nutrition and the other one used in cells subjected to nitrogen stepdown and in nitrogen-fixing filaments. The latter promoter, whose use was NtcA-dependent but HetR-independent, was functional in heterocysts. The N-control transcriptional regulator NtcA was observed to bind in vitro to this promoter. For the sake of comparison, the transcription start points of the nifHDK operon in strain PCC 7120 and binding of NtcA to the nifHDK promoter were also examined.

Ammonium is an important nitrogen source for many microorganisms and plants. Ammonium transporters whose activity can be probed with [14C]methylammonium have been described in several organisms including some cyanobacteria, and amt genes encoding ammonium/methylammonium permeases have been recently identified in yeast, Arabidopsis thaliana, and some bacteria. The unicellular cyanobacterium Synechocystis sp. PCC 6803 exhibited a [14C]methylammonium uptake activity that was inhibited by externally added ammonium. Three putative amt genes that are found in the recently published complete sequence of the chromosome of strain PCC 6803 were inactivated by insertion of antibiotic resistance-encoding gene-cassettes. The corresponding mutant strains were impaired in uptake of [14C]methylammonium. Open reading frame sll0108 (amt1) was responsible for a high affinity uptake activity (Ks for methylammonium, 2.7 microM), whereas open reading frames sll1017 (amt2) and sll0537 (amt3) made minor contributions to uptake at low substrate concentrations. Expression of the three amt genes was higher in nitrogen-starved cells than in cells incubated in the presence of a source of nitrogen (either ammonium or nitrate), but amt1 was expressed at higher levels than the other two amt genes. Transcription of amt1 was found to take place from a promoter bearing the structure of the cyanobacterial promoters activated by the nitrogen control transcription factor, NtcA.

Bacteria secrete small signal molecules into the environment that induce self and neighbour gene expression. This phenomenon, termed quorum sensing, allows cooperative behaviours that increase the fitness of the group. The best-studied signal molecules are the N-acylhomoserine lactones (AHLs), secreted by a growing number of bacterial species including important pathogen species such as Pseudomonas aeruginosa. These molecules have recently been proposed to have properties other than those of signalling, functioning as iron quelants or antibiotics. As the presence of an acylase capable of inactivating long-chain AHLs in Anabaena sp. PCC7120 could constitute a defence mechanism against these molecules, in this work we analyse the effects of different AHLs varying in length and substitutions on the growth and nitrogen metabolism of the cyanobacterium Anabaena sp. PCC7120. All the AHLs tested strongly inhibited nitrogen fixation. The inhibition seems to take place at post-transcriptional level, as no effect on heterocyst differentiation or on the expression of nitrogenase was observed. Moreover, N-(3-oxodecanoyl)-l-homoserine lactone (OC10-HSL) showed a specific cytotoxic effect on this cyanobacterium in the presence of a combined nitrogen source, but the mechanism involved seems to be different from that described so far for tetramic acid derivatives of oxo-substituted AHLs. These results suggest a variety of new unexpected activities for AHLs, at least on cyanobacterial populations.

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 genomewide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N 2 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., This work was supported by the Deutsche Forschungsgemeinschaft program “Sensory and regulatory RNAs in Prokaryotes” SPP1258 Grant HE 2544 4-2; German Federal Ministry of Education and Research Grant 0313921 (to W.R.H.); and by the Ministerio de Ciencia e Innovación Grants BFU2007-60651 (to A.V.) and BFU2010-14821 (to A.M.P.) cofinanced by European Regional Development Fund.

Acyl-acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed. © 2010 Elsevier Ltd., This work was supported by MICINN (PSS-420000-2008-19) and FEDER.

In response to nitrogen deficiency, some cyanobacteria develop heterocysts, a terminally differentiated cell type, specialized for the fixation of atmospheric nitrogen. In Nostocales, this differentiation process is controlled by two major regulators, NtcA and HetR, but additional unknown factors are likely to be involved as well. In the context of a genome-wide search for potential non-coding RNAs, we identified an array of 12 tandem repeats that is transcribed in large amounts when cells enter conditions that trigger cell differentiation and switch to nitrogen fixation. The main accumulating transcript, which we suggest designating nitrogen stress-induced RNA 1 (NsiR1), has properties similar to regulatory non-coding RNAs. In Anabaena sp. PCC 7120, it is about 60 nt in length, has a very distinct predicted secondary structure, and is expressed very early and transiently after nitrogen step-down. Moreover, its expression requires HetR and NtcA and is restricted to cells that are differentiating into heterocysts, clearly placing NsiR1 within the regulon that controls the switch to nitrogen fixation and heterocyst formation. The genomic arrangement of NsiR1, located upstream of hetF, a gene whose product is involved in heterocyst formation, is conserved in all five Nostocales whose genomes are completely sequenced. Additionally, we detected NsiR1 expression in 19 different heterocyst-forming cyanobacteria. Our data suggest that every repeat is a complete transcriptional unit furnished with a cell-type-specific promoter and a Rho-independent terminator, which gives rise to a very high NsiR1 transcript level. NsiR1 is the first known bacterial non-coding RNA that is specifically upregulated in response to nitrogen step-down. © 2010 Elsevier Ltd.

Transcription of the hetC gene, whose product is required for heterocyst differentiation, takes place from a long promoter region that includes the previously described HetR-independent, NtcA-activated promoter producing transcripts with a 5′ end corresponding to position -571 with respect to the translational start site of hetC. Northern blot analysis indicated that the accumulation of hetC transcripts depends on HetR, and a second transcriptional start site located at position -293 that leads to NtcA-dependent, HetR-dependent inducible transcription of hetC was identified. Upon nitrogen stepdown, expression of a PhetC::gfp fusion was transiently induced in specific cells that were differentiating into heterocysts, both when the whole promoter region (containing transcription start points -571 and -293) or a short version (containing only the transcription start point -293) was used. Expression of hetC from the -293 position was delayed in a strain bearing a deleted promoter region lacking sequences upstream from position -570. Such a strain was still able to differentiate functional heterocysts and to grow diazotrophically, although diazotrophic growth was impaired under certain conditions. Similarly, a second, NtcA-dependent, HetR-dependent transcriptional start site was identified at position -454 in the promoter region upstream from the devBCA operon encoding an ABC transport system involved in heterocyst maturation, in which an NtcA-dependent promoter producing transcripts starting at position -704 had been previously noted. Thus, the hetC and devBCA promoter regions exhibit similar tandem promoter arrangements. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The nitrogen-fixing cyanobacterium Nostoc sp. ATCC 29132 was shown to contain two sequence-specific endonucleases. Nsp(29132) I was an isoschizomer of AsuII, and Nsp(29132) II was an isoschizomer of BamHI. Nsp(29132) II was shown to generate ends that could be ligated to those generated by BamHI.

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N2 in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the ntcA gene promoter from Anabaena sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the ntcA gene during heterocyst differentiation was studied in vivo by using an ntcA-gfp translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of ntcA in proheterocysts, but mutation of both sites led to very low, nonlocalized expression. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N2 fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Journal of Bacteriology Vol. 189(10) p.3884-3890, Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N2) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.

Fatty acid desaturases (FAD) play an important role in plant lipid metabolism and they can be found in several subcellular compartments such as the plastids and endoplasmic reticulum. Lipids are critical components of the cell membrane and, as a consequence, they are fundamental for the proper growth and development of all living organisms. We have used sequences from the conserved regions of known omega-3-desaturases to design degenerated oligonucleotides and clone a cDNA encoding a plastidial omega-3 desaturase from sunflower (HaFAD7). From its presumed full-length sequence, we predict that Hafad7 encodes a protein of 443 amino acids with a molecular mass of 50.8 kDa, and that it contains a putative chloroplast transit peptide of 51 amino acids. The predicted hydrophobicity of the protein identifies four potential membrane-spanning regions and, according to the TargetP algorithm, the protein should be targeted to the plastid/chloroplast membrane. RT-PCR analysis of its expression shows the transcript is preferentially expressed in photosynthetically active tissues. Heterologous expression of this protein in the unicellular cyanobacterium Synechocystis sp. PCC 6803 confirmed that the protein produced from this cDNA has omega-3 desaturase activity.