Anabaena Strains Research Articles

ThyD Is a Thylakoid Membrane Protein Influencing Cell Division and Acclimation to High Light in the Multicellular Cyanobacterium Anabaena sp. Strain PCC 7120.

Cyanobacteria developed oxygenic photosynthesis and represent the phylogenetic ancestors of chloroplasts. The model strain Anabaena sp. strain PCC 7120 grows as filaments of communicating cells and can form heterocysts, cells specialized for N2 fixation. In the Anabaena genome, ORF all2390 is annotated as encoding a SulA homolog, but sequence similarity to SulA of model bacteria is insignificant. We generated strains that lacked or overexpressed all2390, both of which showed instances of increased cell size, and observed that purified All2390 protein interfered with the invitro polymerization of FtsZ. Heterocyst frequency diminished by all2390 inactivation and increased by all2390 overexpression. Overexpression retarded the dismantlement of Z-ring structures that determines commitment in the differentiating cells. Thus, All2390 can influence cell division affecting heterocyst differentiation. An All2390-GFP fusion protein localized to the thylakoid membranes including the honeycomb membranes, which harbor photosynthetic complexes, in the heterocyst polar regions. Notably, all2390 expression strongly increased under high light, conditions under which growth of the null mutant is compromised. Thus, All2390 appears essential for adaptation to high light conditions. We named All2390 ThyD to reflect its thylakoidal localization and its dual role in cell division dynamics and acclimation of thylakoid membranes to increased light intensity.

A Four-Step Platform to Optimize Growth Conditions for High-Yield Production of Siderophores in Cyanobacteria.

In response to Iron deprivation and in specific environmental conditions, the cyanobacteria Anabaena flos aquae produce siderophores, iron-chelating molecules that in virtue of their interesting environmental and clinical applications, are recently gaining the interest of the pharmaceutical industry. Yields of siderophore recovery from in vitro producing cyanobacterial cultures are, unfortunately, very low and reach most of the times only analytical quantities. We here propose a four-step experimental pipeline for a rapid and inexpensive identification and optimization of growth parameters influencing, at the transcriptional level, siderophore production in Anabaena flos aquae. The four-steps pipeline consists of: (1) identification of the promoter region of the operon of interest in the genome of Anabaena flos aquae; (2) cloning of the promoter in a recombinant DNA vector, upstream the cDNA coding for the Green Fluorescent Protein (GFP) followed by its stable transformation in Escherichia Coli; (3) identification of the environmental parameters affecting expression of the gene in Escherichia coli and their application to the cultivation of the Anabaena strain; (4) identification of siderophores by the combined use of high-resolution tandem mass spectrometry and molecular networking. This multidisciplinary, sustainable, and green pipeline is amenable to automation and is virtually applicable to any cyanobacteria, or more in general, to any microorganisms.

Gamma (γ)-radiation stress response of the cyanobacterium Anabaena sp. PCC7120: Regulatory role of LexA and photophysiological changes

High radioresistance of the cyanobacterium, Anabaena sp. PCC7120 has been attributed to efficient DNA repair, protein recycling, and oxidative stress management. However, the regulatory network involved in these batteries of responses remains unexplored. In the present study, the role of a global regulator, LexA in modulating gamma (γ)-radiation stress response of Anabaena was investigated. Comparison of the cytosolic proteome profiles upon γ-radiation in recombinant Anabaena strains, AnpAM (vector-control) and AnlexA+ (LexA-overexpressing), revealed 41 differentially accumulated proteins, corresponding to 29 distinct proteins. LexA was found to be involved in the regulation of 27 of the corresponding genes based on the presence of AnLexA-Box, EMSA, and/or qRT-PCR studies. The majority of the regulated genes were found to be involved in C-assimilation either through photosynthesis or C-catabolism and oxidative stress alleviation. Photosynthesis, measured in terms of PSII photophysiological parameters and thylakoid membrane proteome was found to be affected by γ-radiation in both AnpAM and AnlexA+ cells, with LexA affecting them even under control growth conditions. Thus, LexA functioned as one of the transcriptional regulators involved in modulating γ-radiation stress response in Anabaena. This study could pave the way for a deeper understanding of the regulation of γ-radiation-responsive genes in cyanobacteria at large.

OUTLOOKS AND CHALLENGES OF HYDROGEN PRODUCTION BY CYANOBACTERIAL ANABAENA SPECIES

Uncontrolled use of conventional energy sources in the 21st century has increased the temperature of the Earth's surface by 2ºС in the last 1 century. Due to the increasing negative aspects of traditional energy sources, the production of hydrogen from microorganisms has quickly become an object of intense research. However, due to their low concentration of biomass, commercial production of hydrogen (H2) from microorganisms has not yet been realized. In this context, it is important to identify strains of hydrogen producers – cyanobacteria, which are actively used in biotechnology, and to carry out work on increasing the possibilities of hydrogen separation. This review examines the importance of the cyanobacterium Anabaena in hydrogen production and their hydrogen release mechanisms, and shows the main factors affecting H2 release. Also, recent technologies of hydrogen separation are included, as well as works on increasing the amount of hydrogen under the influence of chemical and physical factors. Due to the low ability of Anabaena strains to catalyze hydrogen molecules at present, future research will be limited to genetic engineering technologies. It is only possible to solve the problems arising in the current biohydrogen industry by conducting genetic, technical and metabolic research in parallel. &nbsp

Biochemical Analyses of Ten Cyanobacterial and Microalgal Strains Isolated from Egyptian Habitats, and Screening for Their Potential against Some Selected Phytopathogenic Fungal Strains

Microalgae and cyanobacteria are rich sources of numerous phytochemical compounds with intrinsic antifungal potential. This research aimed to screen the phytochemical compounds and contents, as well as the antioxidant profiles, in eight cyanobacterial and two microalgal strains isolated from soil and brackish water habitats in Egypt. Our study also evaluated their antifungal activities against three phytopathogenic fungi—namely, Pythium ultimum, Fusarium solani, and Botryodiplodia theobromae, which are known to cause severe plant loss. The biochemical compounds were obtained from the cyanobacterial and algal methanolic extracts, and were identified through comparative phytochemical analyses related to the inhibition of the fungal pathogens. Comparative qualitative analyses of alkaloids, steroids, glycosides, and saponins were also carried out. The quantitative phytochemical screening of the cyanobacterial and algal strains investigated revealed the presence of xylanase, glucanase, and chitinase enzymes, along with some bioactive compounds, such as phenolics, flavonoids, proteins, neutral sugars, and carotenoids, which were species-dependent and detected in variable amounts in the extracts. The unicellular green microalgal strain Dunaliella sp. HSSASE13 displayed the highest level of antioxidant activity. However, the highest antifungal activities were shown by the heterocystous cyanobacterial strain Anabaena sp. HSSASE11 (83.94%), followed by Dunaliella sp. HSSASE13 (81.94%) and the non-heterocystous cyanobacterial strain Oscillatoria nigro-viridis HSSASE 15 (63.42%), against the three fungal pathogens B. theobromae, F. solani, and P. ultimum, respectively. Our results indicate that the highest significant and positive correlations of flavonoids (r = 0.854), phenolics (r = 0.785), DPPH scavenging activity (r = 0.876), total proteins (r = 0.808), xylanase activity (r = 0.876), glucanase activity (r = 0.746), and total neutral sugars (r = 0.764), in terms of their antifungal activities, were recorded against F. solani. Conclusively, the cyanobacterial and algal strains tested in the present study can be useful agents for the management and biocontrol of plant-infecting fungal pathogens.

Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria.

Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer-Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria.

Regulatory role of LexA in modulating photosynthetic redox poise and cadmium stress tolerance in the cyanobacterium, Anabaena sp. PCC7120

Strategies developed by organisms to overcome disruption in redox poise of photosynthetic electron transport chain (pETC) are important for its survival under abiotic stress. The process needs to be tightly regulated for optimal functioning. While the redox poising processes are well known in cyanobacteria, understanding of their regulatory network is lacking. Since LexA is one of the known global regulators of stress response in the cyanobacterium Anabaena sp. PCC7120, its role in pETC redox poising was investigated using cadmium (Cd) as an abiotic stressor to disrupt photosynthesis. Assessment of the photosynthetic responses of recombinant Anabaena strains, An lexA + (LexA-overexpressing) and AnpAM (vector control), under unstressed and Cd-stressed conditions using transmission electron microscopy (TEM) and chlorophyll a fluorescence, indicated that some pETC redox poising responses, including PSII photodamage, energy dissipation, PSI photoprotection, and NDH-mediated cyclic electron flow were decreased in An lexA + under unstressed conditions. Disturbance in pETC redox poise during Cd stress observed in Anabaena was accentuated upon overexpression of LexA. The decreased photodamage of PSII and increased photoinhibition of PSI in An lexA + in the presence or absence of Cd stress, correlated well with the changes in pETC complexes observed in blue native (BN)-PAGE and the regulation of over 70 of the 90 pETC component genes by LexA demonstrated through transcript, electromobility shift assay (EMSA), and bioinformatics studies. In a nutshell, LexA has been identified as one of the regulators involved in the streamlining of pETC redox poising responses under normal growth and during abiotic stress through transcriptional regulation of some of the redox-controlled pETC component genes. • Abiotic stresses disrupt photosynthetic redox poise, triggering poising responses. • Poising responses are known in cyanobacteria, but their regulatory network is not. • LexA overexpression in Anabaena reduced these responses, lowering stress tolerance. • In-silico , qRT-PCR and EMSA showed LexA regulation on over 70 photosynthetic genes. • LexA was shown to modulate these responses by regulating some photosynthetic genes.

AzuR From the SmtB/ArsR Family of Transcriptional Repressors Regulates Metallothionein in Anabaena sp. Strain PCC 7120.

Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.

Loading Effects of Aminoclays in Co-Culture of Two Cyanobacterial Microcystis and Anabaena Species as an Algicidal Role

In recent decades, harmful algal blooms (HABs) have been significantly affecting environments, aquatic ecosystems, and human health, as well as damaging economies, especially near rivers and lakes, and in coastal regions. Microcystis and Anabaena are two genera of harmful cyanobacteria that will often predominate during toxic microalgal blooms. In this study, we employ a method for control and mitigation of HABs by microalgal cell instability using different types of aminoclays (ACs). Allelopathic interactions between the two strains of algae are studied in mono-culture, co-culture, and filtrated cell-free medium in the presence of the ACs. The growth of the Anabaena strain is significantly reduced by the cyanobacterial strains in the co-culture media, and both are significantly affected by the Acs’-enhanced algicidal activity. Anabaena sp. KVSF7 shows higher sensitivity against the ACs than does Microcystis sp. KW. In this way, the algicidal activity of ACs is harnessed, the effects of which are in the order of aluminum aminoclay (AlAC) > magnesium aminoclay (MgAC) > calcium aminoclay (CaAC). The ammonium sites in the ACs carry positive charges to induce instability of HABs along with the electrostatic attraction between algal cells and AC. Therefore, the utilization of the algicidal activity of the ACs can effectively reduce HABs, especially on cyanobacterial blooms.

Cyanobacterial amendment boosts plant growth and flower quality in Chrysanthemum through improved nutrient availability

Cultivation of ornamentals requires heavy inputs of chemicals, which can be provided in an environment-friendly manner through cyanobacterial inoculation, facilitating better availability of key macro/micronutrients from the surrounding environment to the host plant and stimulate plant growth. Therefore, the objective of the present investigation was to compare a nursery of Chrysanthemum variety Pusa Aditya grown using potting medium amended with a diazotrophic cyanobacterial strain Anabaena torulosa, with the routine nursery potting medium, for their potential towards improving flower traits, soil fertility and crop productivity. In order to demonstrate the nitrogen savings, the plantlets from such primed nurseries were transplanted into pots, receiving two different doses of nitrogenous fertilizer (full N and 75% N), along with full doses of recommended P and K fertilizers. An increment of 1.8-fold in terms of nitrogen availability in soil was recorded in treatments receiving 75% N along with full doses of recommended P and K fertilizers and cyanobacterium-primed nursery, as compared to all other treatments. An average increase of 35% in the fresh weight of flowers, flower pigmentation as well as early appearance of flower buds was recorded. A significant stimulation of antioxidant and defense enzyme activities in plants, due to cyanobacterium amendment at nursery stage was also recorded. Correlation analyses illustrated a positive association of soil biological attributes (root biofilm formation, soil polysaccharides, glomalin related soil proteins) with most parameters related to plant growth, particularly, reproductive development and flower quality aspects. Principal component analysis illustrated the greater association of these parameters with the treatments involving Anabaena torulosa amended nurseries. It can be concluded that cyanobacterium-amended nurseries can be a useful intervention for deriving robust planting units and leading to beneficial influences on soil nutrient availability, flower quality traits and yield.

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.

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S.elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S.elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

Influence of culture age, ammonium and organic carbon in hydrogen production and nutrient removal by Anabaena sp. in nitrogen-limited cultures

Hydrogen can be generated from cyanobacteria cultivation with light and organic carbon as an energy source. The aim of this study was to investigate the influence of ammonium and glucose concentration, and the culture age on the production of hydrogen and other products, and phosphate and organic carbon removal by Anabaena sp. (UTEX 1448) in batch anaerobic photobioreactors. Our results demonstrated an effect of culture age and ammonium concentration in hydrogen production, an average increase of 4.1 times (90.4 μmol H2 mg Chl a−1 h−1 and 13.2 mmol mg Chl a−1) in the conditions with younger biomass and without ammonium. Culture age also had an effect in phosphate removal, with 92% of removal efficiency, and ethanol production (an average increase of 2.9 times–97 mg L−1), however the optimum conditions were obtained with older biomass. This study demonstrates efficient hydrogen production by this strain of Anabaena sp. fewer researched.

Cyanobacterial Dihydroxyacid Dehydratases Are a Promising Growth Inhibition Target.

Microbes are essential to the global ecosystem, but undesirable microbial growth causes issues ranging from food spoilage and infectious diseases to harmful cyanobacterial blooms. The use of chemicals to control microbial growth has achieved significant success, while specific roles for a majority of essential genes in growth control remain unexplored. Here, we show the growth inhibition of cyanobacterial species by targeting an essential enzyme for the biosynthesis of branched-chain amino acids. Specifically, we report the biochemical, genetic, and structural characterization of dihydroxyacid dehydratase from the model cyanobacterium Synechocystis sp. PCC 6803 (SnDHAD). Our studies suggest that SnDHAD is an oxygen-stable enzyme containing a [2Fe-2S] cluster. Furthermore, we demonstrate that SnDHAD is selectively inhibited in vitro and in vivo by the natural product aspterric acid, which also inhibits the growth of representative bloom-forming Microcystis and Anabaena strains but has minimal effects on microbial pathogens with [4Fe-4S] containing DHADs. This study suggests DHADs as a promising target for the precise growth control of microbes and highlights the exploration of other untargeted essential genes for microbial management.

Features of Anabaena PCC 7120ΔHUP Mutants with Amino Acid Substitutions in Nitrogenase

Mutant strains of the filamentous cyanobacterium Anabaena strain PCC 7120 ΔHup (dc-Q193S and dc-R284H) with amino acid substitutions located in the vicinity of the FeMo cofactor of nitrogenase possess nitrogenase activity with a hydrogen production rate of approximately 18 μmol Н2/(mg h), which is ~30% lower than that of the parental strain ΔHup. The photosynthetic activity of mutants is also reduced. Unlike the parental strain ΔHup, the dc-Q193S mutant shows a lower temperature optimum for hydrogen photoproduction. This difference is probably due to the lowered filament strength (fragmentation). Hydrogen photoproduction in mutants does not significantly differ from that of the parental strain in relation to the activation energy, light saturation constants (41–62 μmol quanta /(m2 s)), and acetylene-induced inhibition. However, in contrast to the parental strain, hydrogen photoproduction in the mutant strains is not inhibited by molecular nitrogen, i.e., amino acid substitutions cause significant changes in the reaction requiring eight electrons (N2 fixation). The possibility to use nitrogen or atmospheric air instead of argon in the hydrogen production is promising from the practical point of view, though reduced activity and increased fragility of filaments in the studied mutants limit the possibility of their practical use.

Study of the effect of nitrogen-fxing cyanobacteria on the growth rate of the Strawberry Sunrise T-4 strawberry variety

The article presents the results of the study of nitrogenase activity of five strains of cyanobacteriafrom the collection of phototrophic microorganisms of Al-Farabi Kazakh National University and their effect on the growth of strawberries. Nitrogen-fixing cyanobacteria were studied by strains Anabaena variabilis R-I-5, Anabaena sp. 7912, Anabaena sp. Z-1, Nostoc calsicola RI-3, Nostoc sp. S-2, and accordingto the results obtained high activity of nitrogenase in the strain Anabaena variabilis R-I-5. In the studyof the effect of cells of different concentrations of the strain Anabaena variabilis R-I-5 on the growth ofstrawberry Strawberry Sunrise T-4, it was found that its suspension of 24x106 cells/ml and 48x106 cell/ml had a positive effect on strawberry growth. In addition, an increase in the number of strawberry rootswas observed in the suspension of cyanobacterial biomass 24x106 cell/ml. An increase in the number ofstrawberry leaves, root length, stem height and a significantly higher yield of dry biomass of strawberrieswere observed in the suspension of the study in the amount of 48x106 cell/ml. The optimal cell count ofthe nitrogen-fixing cyanobacterium Anabaena variabilis R-I-5 suspension, which has a positive effect onthe growth of strawberry plants, was 48x106 cell/ml. The obtained results allow to use the nitrogen fixingstrain Anabaena variabilis R-I-5 for obtaining biological products in agrobiotechnology.Key words: Nitrogen-fixing cyanobacteria strains, grow of Strawberry Sunrise T-4 variety, molecularnitrogen absorption in air.

Regulation by FurC in Anabaena Links the Oxidative Stress Response to Photosynthetic Metabolism.

The FUR (Ferric Uptake Regulator) family in Anabaena sp. PCC 7120 consists of three paralogs named FurA (Fur), FurB (Zur) and FurC (PerR). furC seems to be an essential gene in the filamentous nitrogen-fixing strain Anabaena sp. PCC 7120, suggesting that it plays a fundamental role in this organism. In order to better understand the functions of FurC in Anabaena, the phenotype of a derivative strain that overexpresses this regulator (EB2770FurC) has been characterized. The furC-overexpressing variant presented alterations in growth rate, morphology and ultrastructure, as well as higher sensitivity to peroxide than Anabaena sp. PCC 7120. Interestingly, the overexpression of furC led to reduced photosynthetic O2 evolution, increased respiratory activity, and had a significant influence in the composition and efficiency of both photosystems. Comparative transcriptional analyses, together with electrophoretic mobility shift assays allowed the identification of different genes directly controlled by FurC, and involved in processes not previously related to PerR proteins, such as the cell division gene ftsZ and the major thylakoid membrane protease ftsH. The rise in the transcription of ftsH in EB2770FurC cells correlated with reduced levels of the D1 protein, which is involved in the PSII repair cycle. Deregulation of the oxidative stress response in EB2770FurC cells led to the identification of novel FurC targets involved in the response to H2O2 through different mechanisms. These results, together with the effect of furC overexpression on the composition, stability and efficiency of the photosynthetic machinery of Anabaena, disclose novel links between PerR proteins, cell division and photosynthesis in filamentous cyanobacteria.

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.

Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine.

Arginine participates widely in metabolic processes. The heterocyst-forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N-terminal α/β propeller domain and a unique C-terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N-terminal domain and ornithine cyclodeaminase activity of the C-terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, β-aspartyl-arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE-PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C-terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.

Anaerobic butanol production driven by oxygen-evolving photosynthesis using the heterocyst-forming multicellular cyanobacterium Anabaena sp. PCC 7120.

Cyanobacteria are oxygen-evolving photosynthetic bacteria. Established genetic manipulation methods and recently developed gene-regulation tools have enabled the photosynthetic conversion of carbon dioxide to biofuels and valuable chemicals in cyanobacteria, especially in unicellular cyanobacteria. However, the oxygen sensitivity of enzyme(s) introduced into cyanobacteria hampers productivity in some cases. Anabaena sp. PCC 7120 is a filamentous cyanobacterium consisting of a few hundred of vegetative cells, which perform oxygenic photosynthesis. Upon nitrogen deprivation, heterocysts, which are specialized cells for nitrogen fixation, are differentiated from vegetative cells at semiregular intervals. The micro-oxic environment within heterocysts protects oxygen-labile nitrogenase from oxygen. This study aimed to repurpose the heterocyst as a host for the production of chemicals with oxygen-sensitive enzymes under photosynthetic conditions. Herein, Anabaena strains expressing enzymes of 1-butanol synthetic pathway from the anaerobe Clostridium acetobutylicum within heterocysts were created. A strain that expressed a highly oxygen-sensitive Bcd/EtfAB complex produced 1-butanol even under photosynthetic conditions. Furthermore, the 1-butanol production per heterocyst cell of a butanol-producing Anabaena strain was fivefold higher than that per cell of unicellular cyanobacterium with the same set of 1-butanol synthetic pathway genes. Thus, our study showed the usefulness of Anabaena heterocysts as a chassis for anaerobic production driven by oxygen-evolving photosynthesis.