Emerging nitrogen-fixing cyanobacteria for sustainable cotton cultivation.

Amid growing environmental concerns and the imperative for sustainable agricultural practices, this study examines the potential of nitrogen-fixing cyanobacteria as biofertilizers, particularly in cotton cultivation. The reliance on synthetic nitrogen fertilizers (SNFs), prevalent in modern agriculture, poses significant environmental challenges, including greenhouse gas emissions and water system contamination. This research aims to shift this paradigm by exploring the capacity of cyanobacteria as a natural and sustainable alternative. Utilizing advanced metabarcoding methods to analyze the 16S rRNA gene, we conducted a comprehensive assessment of soil bacterial communities within cotton fields. This study focused on evaluating the diversity, structure, taxonomic composition, and potential functional characteristics of these communities. Emphasis was placed on the isolation of native N2-fixing cyanobacteria strains rom cotton soils, and their subsequent effects on cotton growth. Results from our study demonstrate significant plant growth-promoting (PGP) activities, measured as N2 fixation, production of Phytohormones, Fe solubilization and biofertilization potential of five isolated cyanobacterial strains, underscoring their efficacy in cotton. These findings suggest a viable pathway for replacing chemical-synthetic nitrogen fertilizers with natural, organic alternatives. The reintegration of these beneficial species into agricultural ecosystems can enhance crop growth while fostering a balanced microbial environment, thus contributing to the broader goals of global sustainable agriculture.

Phylogenetic and functional analysis of cyanobacterial Cytochrome c6-like proteins.

All known photosynthetic cyanobacteria carry a cytochrome c 6 protein that acts transferring electrons from cytochrome b 6 f complex to photosystem I, in photosynthesis, or cytochrome c oxidase, in respiration. In most of the cyanobacteria, at least one homologue to cytochrome c 6 is found, the so-called cytochrome c 6B or cytochrome c 6C. However, the function of these cytochrome c 6-like proteins is still unknown. Recently, it has been proposed a common origin of these proteins as well as the reclassification of the cytochrome c 6C group as c 6B, renaming the new joint group as cytochrome c 6BC. Another homologue to cytochrome c 6 has not been classified yet, the formerly called cytochrome c 6-3, which is present in the heterocyst-forming filamentous cyanobacteria Nostoc sp. PCC 7119. In this work, we propose the inclusion of this group as an independent group in the genealogy of cytochrome c 6-like proteins with significant differences from cytochrome c 6 and cytochrome c 6BC, with the proposed name cytochrome c 6D. To support this proposal, new data about phylogeny, genome localisation and functional properties of cytochrome c 6-like proteins is provided. Also, we have analysed the interaction of cytochrome c 6-like proteins with cytochrome f by isothermal titration calorimetry and by molecular docking, concluding that c 6-like proteins could interact with cytochrome b 6 f complex in a similar fashion as cytochrome c 6. Finally, we have analysed the reactivity of cytochrome c 6-like proteins with membranes enriched in terminal oxidases of cyanobacteria by oxygen uptake experiments, concluding that cytochrome c 6D is able to react with the specific copper-oxidase of the heterocysts, the cytochrome c oxidase 2.

Symbiosis between cyanobacteria and plants: from molecular studies to agronomic applications.

Nitrogen-fixing cyanobacteria from the order Nostocales are able to establish symbiotic relationships with diverse plant species. They are promiscuous symbionts, as the same strain of cyanobacterium is able to form symbiotic biological nitrogen-fixing relationships with different plants species. This review will focus on the different types of cyanobacterial-plant associations, both endophytic and epiphytic, and provide insights from a structural viewpoint, as well as our current understanding of the mechanisms involved in the symbiotic crosstalk. In all these symbioses, the benefit for the plant is clear; it obtains from the cyanobacterium fixed nitrogen and other bioactive compounds, such as phytohormones, polysaccharides, siderophores, or vitamins, leading to enhanced plant growth and productivity. Additionally, there is increasing use of different cyanobacterial species as bio-inoculants for biological nitrogen fixation to improve soil fertility and crop production, thus providing an eco-friendly, alternative, and sustainable approach to reduce the over-reliance on synthetic chemical fertilizers.

Plant Growth-Promoting Rhizobacteria Improve Rice Response to Climate Change Conditions.

Rice is one of the most important crops in the world and is considered a strategic crop for food security. Furthermore, the excessive use of chemical fertilizers to obtain high yields causes environmental problems. A sustainable alternative includes taking advantage of beneficial bacteria that promote plant growth. Here, we investigate the effect of five bacterial biofertilizers from halophytes on growth, and we investigate photosynthetic efficiency in rice plants grown under saline conditions (0 and 85 mmol L-1 NaCl) and future climate change scenarios, including increased CO2 concentrations and temperature (400/700 ppm and 25/+4 °C, respectively). Biofertilizers 1-4 increased growth by 9-64% in plants grown with and without salt in both CO2- temperature combinations, although there was no significant positive effect on the net photosynthetic rate of rice plants. In general, biofertilizer 1 was the most effective at 400 ppm CO2 and at 700 ppm CO2 +4 °C in the absence of salt. Inocula 1-5 also stimulated plant length at high CO2 levels without salt. Finally, the positive effect of biofertilization was attenuated in the plants grown under the interaction between salt and high CO2. This highlights the significance of studying biofertilization under stress interaction to establish the real potential of biofertilizers in the context of climate change conditions.

Quantitative Proteomics at Early Stages of the Symbiotic Interaction Between Oryza sativa and Nostoc punctiforme Reveals Novel Proteins Involved in the Symbiotic Crosstalk.

Symbiosis between cyanobacteria and plants is considered pivotal for biological nitrogen deposition in terrestrial ecosystems. Despite extensive knowledge of the ecology of plant-cyanobacterium symbioses, little is known about the molecular mechanisms involved in recognition between partners. Here, we conducted a quantitative sequential window acquisition of all theoretical fragment ion spectra mass spectrometry pipeline to analyze protein changes in Oryza sativa and Nostoc punctiforme during early events of symbiosis. We found differentially expressed proteins in both organisms linked to several biological functions, including signal transduction, adhesion, defense-related proteins and cell wall modification. In N. punctiforme we found increased expression of 62 proteins that have been previously described in other Nostoc-plant symbioses, reinforcing the robustness of our study. Our findings reveal new proteins activated in the early stages of the Nostoc-Oryza symbiosis that might be important for the recognition between the plant and the host. Oryza mutants in genes in the common symbiosis signaling pathway (CSSP) show reduced colonization efficiency, providing first insights on the involvement of the CSSP for the accommodation of N. punctiforme inside the plant cells. This information may have long-term implications for a greater understanding of the symbiotic interaction between Nostoc and land plants.

Endophytic Colonization of Rice (Oryza sativa L.) by the Symbiotic Strain Nostoc punctiforme PCC 73102.

Cyanobacteria are phototrophic microorganisms able to establish nitrogen-fixing symbiotic associations with representatives of all four of the major phylogenetic divisions of terrestrial plants. Despite increasing knowledge on the beneficial effects of cyanobacteria in rice fields, the information about the interaction between these microorganisms and rice at the molecular and structural levels is still limited. We have used the model nitrogen-fixing cyanobacterium Nostoc punctiforme to promote a long-term stable endophytic association with rice. Inoculation with this strain of hydroponic cultures of rice produces a fast adherence of the cyanobacterium to rice roots. At longer times, cyanobacterial growth in the proximity of the roots increased until reaching a plateau. This latter phase coincides with the intracellular colonization of the root epidermis and exodermis. Structural analysis of the roots revealed that the cyanobacterium use an apoplastic route to colonize the plant cells. Moreover, plant roots inoculated with N. punctiforme show both the presence of heterocysts and nitrogenase activity, resulting in the promotion of plant growth under nitrogen deficiency, thus providing benefits for the plant.

Cytochrome c6 is the main respiratory and photosynthetic soluble electron donor in heterocysts of the cyanobacterium Anabaena sp. PCC 7120.

Cytochrome c6 is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b6-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c6 and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c6 is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c6 has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c6 deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c6 plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.

Management of enzyme diversity in high-performance cellulolytic cocktails.

BACKGROUND: Modern biorefineries require enzymatic cocktails of improved efficiency to generate fermentable sugars from lignocellulosic biomass. Cellulolytic fungi, among other microorganisms, have demonstrated the highest potential in terms of enzymatic productivity, complexity and efficiency. On the other hand, under cellulolytic-inducing conditions, they often produce a considerable diversity of carbohydrate-active enzymes which allow them to adapt to changing environmental conditions. However, industrial conditions are fixed and adjusted to the optimum of the whole cocktail, resulting in underperformance of individual enzymes. RESULTS: One of these cellulolytic cocktails from Myceliophthora thermophila has been analyzed here by means of LC-MS/MS. Pure GH6 family members detected have been characterized, confirming previous studies, and added to whole cocktails to compare their contribution in the hydrolysis of industrial substrates. Finally, independent deletions of two GH6 family members, as an example of the enzymatic diversity management, led to the development of a strain producing a more efficient cellulolytic cocktail. CONCLUSIONS: These data indicate that the deletion of noncontributive cellulases (here EG VI) can increase the cellulolytic efficiency of the cocktail, validating the management of cellulase diversity as a strategy to obtain improved fungal cellulolytic cocktails.

Cyt c6-3: A New Isoform of Photosynthetic Cyt c6 Exclusive to Heterocyst-Forming Cyanobacteria.

All known cyanobacteria contain Cyt c6, a small soluble electron carrier protein whose main function is to transfer electrons from the Cyt b6f complex to PSI, although it is also involved in respiration. We have previously described a second isoform of this protein, the Cyt c6-like, whose function remains unknown. Here we describe a third isoform of Cyt c6 (here called Cytc6-3), which is only found in heterocyst-forming filamentous cyanobacteria. Cyt c6-3 is expressed in vegetative cells but is specifically repressed in heterocysts cells under diazotrophic growth conditions. Although there is a close structural similarity between Cyt c6-3 and Cyt c6 related to the general protein folding, Cyt c6-3 presents differential electrostatic surface features as compared with Cyt c6, its expression is not copper dependent and has a low reactivity towards PSI. According to the different expression pattern, functional reactivity and structural properties, Cyt c6-3 has to play an as yet to be defined regulatory role related to heterocyst differentiation.

Recalcitrant carbohydrates after enzymatic hydrolysis of pretreated lignocellulosic biomass.

BACKGROUND: To reduce the cost of the enzymes for the hydrolysis of lignocellulosic biomass, two main strategies have been followed: one, the reduction of enzyme dosing by the use of more efficient and stable enzymatic cocktails; another, to include accessory enzymes in the cocktails to increase yields by reducing the recalcitrant carbohydrate fraction remaining at the end of the process. To guide this second strategy, we have explored the chemical bond composition of different fractions of recalcitrant carbohydrates after enzymatic hydrolysis. RESULTS: Two lignocellulosic feedstocks of relevance for the biofuels industry have been analyzed, corn stover and sugarcane straw. On comparing the composition of chemical bonds of the starting pretreated material with samples after standard and forced hydrolysis (with enzyme overdosing), we obtained similar sugar and chemical bond composition. CONCLUSIONS: This suggests that the current enzymatic cocktails bear the set of enzymes needed to hydrolyze these feedstocks. From our point of view, the results show the need for a parallel fine-tuning of the enzymatic cocktails with the pretreatment process to maximize sugar release yield.

Interaction of photosystem I from Phaeodactylum tricornutum with plastocyanins as compared with its native cytochrome c6: Reunion with a lost donor.

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself.

Photosystem I reduction in diatoms: as complex as the green lineage systems but less efficient.

Diatoms occupy a key branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. Here, the electron transfer reaction mechanism from cytochrome c6 to photosystem I from the diatom Phaeodactylum tricornutum has been analyzed by laser-flash absorption spectroscopy. Kinetic traces of photosystem I reduction fit to biphasic curves, the analysis of the observed rate constants indicating that electron transfer occurs in a cytochrome c6/photosystem I transient complex, which undergoes a reorganization process from the initial encounter complex to the optimized final configuration. The mild ionic strength dependence of the rate constants makes evident the relatively weak electrostatically attractive nature of the interaction. Taken together, these results indicate that the "red" Phaeodactylum system is less efficient than "green" systems, both in the formation of the properly arranged (cytochrome c6/photosystem I) complex and in the electron transfer itself. The results obtained from cross-reactions with cytochrome c6 and photosystem I from cyanobacteria, green algae, and plants shed light on the different evolutionary pathway of the electron transfer to photosystem I in diatoms with regard to the way that it evolved in higher plants.

Cytochrome c6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119.

Most organisms performing oxygenic photosynthesis contain either cytochrome c(6) or plastocyanin, or both, to transfer electrons from cytochrome b(6)-f to photosystem I. Even though plastocyanin has superseded cytochrome c(6) along evolution, plants contain a modified cytochrome c(6), the so called cytochrome c(6A), whose function still remains unknown. In this article, we describe a second cytochrome c(6) (the so called cytochrome c(6)-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c(6A) than to cyanobacterial cytochrome c(6). In this article, we conclude that the cytochrome c(6)-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c(6) and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c(6) and plastocyanin. In any way, the Cyt c(6)-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.

A novel alpha-amylase from the cyanobacterium Nostoc sp. PCC 7119.

Little information is yet available on the alpha-amylases of cyanobacteria. Here, the presence of an alpha-amylase in the cyanobacterium Nostoc sp. PCC 7119 is first demonstrated. A gene (amy1) encoding a cytoplasmic alpha-amylase (Amy1) protein has been identified, cloned, and overexpressed in Escherichia coli cells. The recombinant protein is a 56.7-kDa monomer, which has been purified to electrophoretic homogeneity by affinity chromatography. The substrate specificity and end product analyses confirm that it is a calcium-dependent alpha-amylase enzyme, which exhibits its maximum activity at 31 degrees C and at pH between 6.5 and 7.5. The Amy1 protein breaks down mainly starch, is also able to cleave glycogen and dextrin, and exhibits no activity against xylan or pullulan. So the enzyme cannot efficiently attack the maltodextrins with degrees of polymerization below that of maltooctaose. Maltotriose, maltose, and maltotetraose are the major products of the enzymatic reaction with starch as substrate. The enzyme shows a very high turnover number against soluble potato starch (3,420 +/- 270 s(-1)), as compared with other alpha-amylases reported in the literature. The high catalytic efficiency and relatively low optimum temperature of the Nostoc Amy1 protein make this previously unexplored group of cyanobacterial enzymes of great interest for further physiological studies and industrial applications.

Gated electron transfer of cytochrome c6 at biomimetic interfaces: a time-resolved SERR study.

The electron shuttle heme protein Cyt-c(6) from the photosynthetic cyanobacterium Nostoc sp. PCC 7119 was immobilized on nanostructured Ag electrodes coated with SAMs that mimic different possible interactions with its natural reaction partner PSI. The structure, redox potential, and electron-transfer dynamics of the SAM-Cyt-c(6) complexes were investigated by TR-SERR spectroelectrochemistry. It is shown that the heterogeneous electron-transfer process is gated both in electrostatic and hydrophobic-hydrophilic complexes. At long tunneling distances, the reaction rate is controlled by the tunneling probability, while at shorter distances or higher driving forces, protein dynamics becomes the rate-limiting event.

Detoxification of superoxide without production of H2O2: antioxidant activity of superoxide reductase complexed with ferrocyanide.

The superoxide radical O(2)(-.) is a toxic by-product of oxygen metabolism. Two O(2)(-.) detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H2O2 as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe(2+) (N-His)(4) (S-Cys)] pentacoordination, was shown to have the ability to form a complex with the organometallic compound ferrocyanide. Here, we have investigated in detail the reactivity of the SOR-ferrocyanide complex with O(2)(-.) by pulse and gamma-ray radiolysis, infrared, and UV-visible spectroscopies. The complex reacts very efficiently with O(2)(-.). However, the presence of the ferrocyanide adduct markedly modifies the reaction mechanism of SOR, with the formation of transient intermediates different from those observed for SOR alone. A one-electron redox chemistry appears to be carried out by the ferrocyanide moiety of the complex, whereas the SOR iron site remains in the reduced state. Surprisingly, the toxic H2O2 species is no longer the reaction product. Accordingly, in vivo experiments showed that formation of the SOR-ferrocyanide complex increased the antioxidant capabilities of SOR expressed in an Escherichia coli sodA sodB recA mutant strain. Altogether, these data describe an unprecedented O(2)(-.) detoxification activity, catalyzed by the SOR-ferrocyanide complex, which does not conduct to the production of the toxic H2O2 species.

A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase.

Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate that the accessibility and electrostatic charge of the active site do not differ in a significant way among the three proteins, but human cytochrome c exhibits some intriguing differences when interacting with cytochrome c oxidase that could be related to the amino acid changes underwent by the latter along evolution.

Laser flash-induced kinetic analysis of cytochrome f oxidation by wild-type and mutant plastocyanin from the cyanobacterium Nostoc sp. PCC 7119.

Oxidation of the soluble, truncated form of cytochrome f by wild-type and mutant species of plastocyanin has been analyzed by laser flash absorption spectroscopy in the cyanobacterium Nostoc (formerly, Anabaena) sp. PCC 7119. At low ionic strengths, the apparent electron transfer rate constant of cytochrome f oxidation by wild-type plastocyanin is 1.34 x 10(4) s(-)(1), a value much larger than those determined for the same proteins from other organisms. Upon site-directed mutagenesis of specific residues at the plastocyanin interaction area, the rate constant decreases in all cases yet to varying extents. The only exception is the D54K variant, which exhibits a higher reactivity toward cytochrome f. In most cases, the reaction rate constant decreases monotonically with an increase in ionic strength. The observed changes in the reaction mechanism and rate constants are in agreement with the location of the mutated residues at the interface area, as well as with the peculiar orientation of the two partners within the Nostoc plastocyanin-cytochrome f transient complex, whose NMR structure has been determined recently. Furthermore, the experimental data herein reported match well the kinetic behavior exhibited by the same set of plastocyanin mutants when acting as donors of electrons to photosystem I [Molina-Heredia, F. P., et al. (2001) J. Biol. Chem. 276, 601-605], thus indicating that the copper protein uses the same surface areas-one hydrophobic and the other electrostatic-to interact with both cytochrome f and photosystem I.

NMR analysis of the transient complex between membrane photosystem I and soluble cytochrome c6.

A structural analysis of the surface areas of cytochrome c(6), responsible for the transient interaction with photosystem I, was performed by NMR transverse relaxation-optimized spectroscopy. The hemeprotein was titrated by adding increasing amounts of the chlorophyllic photosystem, and the NMR spectra of the free and bound protein were analyzed in a comparative way. The NMR signals of cytochrome c(6) residues located at the hydrophobic and electrostatic patches, which both surround the heme cleft, were specifically modified by binding. The backbones of internal residues close to the hydrophobic patch of cytochrome c(6) were also affected, a fact that is ascribed to the conformational changes taking place inside the hemeprotein when interacting with photosystem I. To the best of our knowledge, this is the first structural analysis by NMR spectroscopy of a transient complex between soluble and membrane proteins.

Structure of superoxide reductase bound to ferrocyanide and active site expansion upon X-ray-induced photo-reduction.

Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-). SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 A resolution, respectively. The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge. The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays. The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site.