The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Physicochemical behavioral changes in consort with nitrogen metabolism of cyanobacterium Anabaena PCC 7120 under arsenite regimes.

The present study was undertaken to investigate the arsenite (As III)-induced changes in the diazotrophic cyanobacterium Anabaena PCC 7120. It was observed that the growth of cyanobacterial decreased with increase in As (III) concentration. The cells exposed to As (III) showed morphological variation (deformity) due to the formation of deeper constrictions in vegetative cells. Strain showed increased heterocyst differentiation (1.6-fold higher) whereas decreased nitrogenase activity at the concentration of 40 ppm As (III). The activities of NR, NiR, urease and GS decreased with increase in As (III) concentrations and attained their minimum levels at 40 ppm of As (III). The Ca2+-dependent ATPase activity increased with increase in As (III) concentration and attained its about 2.72-fold higher level at 40 ppm of As (III). In contrast, sharp decline in Mg2+-dependent ATPase activity (28%) was recorded at 1 ppm of As (III) over untreated control. The rates of photosynthetic O2 evolution and respiration decreased with increase in As (III) concentration and attained its minimal level at 40 ppm of As (III). Therefore, this study highlighted arsenite regimes efficiently correlated with behavioral changes in consort with strain.

ß-N-Methylamino-L-Alanine (BMAA) Causes Severe Stress in Nostoc sp. PCC 7120 Cells under Diazotrophic Conditions: A Proteomic Study.

Non-proteinogenic neurotoxic amino acid ß-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms' ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA's action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected-due to their notable expression differences-for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and ß (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes; BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.

Application of Nitrate, Ammonium, or Urea Changes the Concentrations of Ureides, Urea, Amino Acids and Other Metabolites in Xylem Sap and in the Organs of Soybean Plants (Glycine max (L.) Merr.).

Soybean (Glycine max (L.) Merr.) plants form root nodules and fix atmospheric dinitrogen, while also utilizing the combined nitrogen absorbed from roots. In this study, nodulated soybean plants were supplied with 5 mM N nitrate, ammonium, or urea for 3 days, and the changes in metabolite concentrations in the xylem sap and each organ were analyzed. The ureide concentration in the xylem sap was the highest in the control plants that were supplied with an N-free nutrient solution, but nitrate and asparagine were the principal compounds in the xylem sap with nitrate treatment. The metabolite concentrations in both the xylem sap and each organ were similar between the ammonium and urea treatments. Considerable amounts of urea were present in the xylem sap and all the organs among all the treatments. Positive correlations were observed between the ureides and urea concentrations in the xylem sap as well as in the roots and leaves, although no correlations were observed between the urea and arginine concentrations, suggesting that urea may have originated from ureide degradation in soybean plants, possibly in the roots. This is the first finding of the possibility of ureide degradation to urea in the underground organs of soybean plants.

The influence of ethylene, gibberellins and brassinosteroids on energy and nitrogen-fixation metabolites in nodule tissue.

Legume nodules are a unique plant organ that contain nitrogen-fixing rhizobial bacteria. For this interaction to be mutually beneficial, plant and bacterial metabolism must be precisely co-ordinated. Plant hormones are known to play essential roles during the establishment of legume-rhizobial symbioses but their role in subsequent nodule metabolism has not been explored in any depth. The plant hormones brassinosteroids, ethylene and gibberellins influence legume infection, nodule number and in some cases nodule function. In this paper, the influence of these hormones on nodule metabolism was examined in a series of well characterised pea mutants with altered hormone biosynthesis or response. A targeted set of metabolites involved in nutrient exchange and nitrogen fixation was examined in nodule tissue of mutant and wild type plants. Gibberellin-deficiency had a major negative impact on the level of several major dicarboxylates supplied to rhizobia by the plant and also led to a significant deficit in the amino acids involved in glutamine-aspartate transamination, consistent with the limited bacteroid development and low fixation rate of gibberellin-deficient na mutant nodules. In contrast, no major effects of brassinosteroid-deficiency or ethylene-insensitivity on the key metabolites in these pathways were found. Therefore, although all three hormones influence infection and nodule number, only gibberellin is important for the establishment of a functional nodule metabolome.

Impact of pesticides in properties of Bradyrhizobium spp. and in the symbiotic performance with soybean.

Soybean [Glycine max (L.) Merr.] has great economic and nutritional importance mainly due to its high protein content. All plant's N needs can be met by the symbiosis with elite Bradyrhizobium strains applied as inoculants to the seeds at sowing time; however, the increasing use of pesticides in seed treatments can impair the contribution of the biological nitrogen fixation. In this study, we report decreases in cell survival of two strains, B. japonicum SEMIA 5079 and B. elkanii SEMIA 587 in seeds inoculated and treated with StandakTop™, composed of the fungicides pyraclostrobin and thiophanate-methyl and the insecticide fipronil, the pesticides most used in soybean seed treatment in several countries. Cell death was enhanced with the time of exposure to the pesticides, and B. elkanii was less tolerant, with almost no detectable viable cells after 15 days. Change in colony morphology with smaller colonies was observed in the presence of the pesticides, being more drastic with the time of exposure, and attributed to an adaptive response towards survival in the presence of the abiotic stress. However, morphological changes were reversible after elimination of the stressing agent and symbiotic performance under controlled greenhouse conditions was similar between strains that had been or not exposed to the pesticides. In addition, no changes in DNA profiles (BOX-PCR) of both strains were observed after the contact with the pesticides. In two field experiments, impacting effects of the pesticides were observed mainly on the total N accumulated in grains of plants relying on both N2-fixation and N-fertilizer. Our data indicate that StandakTop® affects parameters never reported before, including colony morphology of Bradyrhizobium spp. and N metabolism and/or N remobilization to soybean grains.

Mechanisms of Rice Endophytic Bradyrhizobial Cell Differentiation and Its Role in Nitrogen Fixation.

Bradyrhizobium sp. strain SUTN9-2 is a symbiotic and endophytic diazotrophic bacterium found in legume and rice plants and has the potential to promote growth. The present results revealed that SUTN9-2 underwent cell enlargement, increased its DNA content, and efficiently performed nitrogen fixation in response to rice extract. Some factors in rice extract induced the expression of cell cycle and nitrogen fixation genes. According to differentially expressed genes (DEGs) from the transcriptomic analysis, SUTN9-2 was affected by rice extract and the deletion of the bclA gene. The up-regulated DEGs encoding a class of oxidoreductases, which act with oxygen atoms and may have a role in controlling oxygen at an appropriate level for nitrogenase activity, followed by GroESL chaperonins are required for the function of nitrogenase. These results indicate that following its exposure to rice extract, nitrogen fixation by SUTN9-2 is induced by the collective effects of GroESL and oxidoreductases. The expression of the sensitivity to antimicrobial peptides transporter (sapDF) was also up-regulated, resulting in cell differentiation, even when bclA (sapDF) was mutated. This result implies similarities in the production of defensin-like antimicrobial peptides (DEFs) by rice and nodule-specific cysteine-rich (NCR) peptides in legume plants, which affect bacterial cell differentiation.

The First Proteomics Study of Nostoc sp. PCC 7120 Exposed to Cyanotoxin BMAA under Nitrogen Starvation.

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostocpunctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

Chlorpyrifos inhibits nitrogen fixation in rice-vegetated soil containing Pseudomonas stutzeri A1501.

Chlorpyrifos, a common organophosphorus pesticide, is widely used for agricultural pest control and can inhibit nitrogen-fixing bacteria biomass in paddy. In this study, the additions of chlorpyrifos (1 and 8 mg kg-1) to soil, with or without Pseudomonas stutzeri A1501, resulted in a significant decrease in nitrogen fixation, despite insignificant effects on the abundances of P. stutzeri A1501 and bacteria in soil. Toxic effect of chlorpyrifos on P. stutzeri A1501 nitrogenase activity in medium was also observed, accompanied by a significant reduction in the expression of nitrogen-fixing related genes (nifA and nifH). Furthermore, rhizosphere colonization and biofilm formation by P. stutzeri A1501 were repressed by chlorpyrifos, leading to decreased nitrogenase activity in the rhizosphere. Biofilm formation in medium was inhibited by bacterial hyperkinesis and reduction of extracellular polymeric substance, including exopolysaccharides and proteins. Together, these findings showed that chlorpyrifos-induced production of reactive oxygen species (ROS) which was directly responsible for reduced nitrogenase activity in the medium, soil, and rhizosphere by inhibiting the expressions of nitrogen-fixing related genes. Furthermore, the inhibition of biofilm formation by chlorpyrifos or ROS likely aggravated the reduction in rhizospherere nitrogenase activity. These findings provide potentially valuable insights into the toxicity of chlorpyrifos on nitrogen-fixing bacteria and its mechanisms. Furthermore, for sustainable rice production, it is necessary to evaluate whether other pesticides affect nitrogen fixation and select pesticides that do not inhibit nitrogen fixation.

Enhancement of soybean nodulation by seed treatment with non-thermal plasmas.

Soybean (Glycine max (L.) Merrill) is one of the most important crops worldwide providing dietary protein and vegetable oil. Most of the nitrogen required by the crop is supplied through biological N2 fixation. Non-thermal plasma is a fast, economical, and environmental-friendly technology that can improve seed quality, plant growth, and crop yield. Soybean seeds were exposed to a dielectric barrier discharge plasma operating at atmospheric pressure air with superimposed flows of O2 or N2 as carrying gases. An arrangement of a thin phenolic sheet covered by polyester films was employed as an insulating barrier. We focused on the ability of plasma to improve soybean nodulation and biological nitrogen fixation. The total number of nodules and their weight were significantly higher in plants grown from treated seeds than in control. Plasma treatments incremented 1.6 fold the nitrogenase activity in nodules, while leghaemoglobin content was increased two times, indicating that nodules were fixing nitrogen more actively than control. Accordingly, the nitrogen content in nodules and the aerial part of plants increased by 64% and 23%, respectively. Our results were supported by biometrical parameters. The results suggested that different mechanisms are involved in soybean nodulation improvement. Therefore, the root contents of isoflavonoids, glutathione, auxin and cytokinin, and expansin (GmEXP1) gene expression were determined. We consider this emerging technology is a suitable pre-sowing seed treatment.

Dissolved organic matter stimulates N2 fixation and nifH gene expression in Trichodesmium.

Mixotrophy, the combination of heterotrophic and autotrophic nutrition modes, is emerging as the rule rather than the exception in marine photosynthetic plankton. Trichodesmium, a prominent diazotroph ubiquitous in the (sub)tropical oceans, is generally considered to obtain energy via autotrophy. While the ability of Trichodesmium to use dissolved organic phosphorus when deprived of inorganic phosphorus sources is well known, the extent to which this important cyanobacterium may benefit from other dissolved organic matter (DOM) resources is unknown. Here we provide evidence of carbon-, nitrogen- and phosphorus-rich DOM molecules enhancing N2 fixation rates and nifH gene expression in natural Trichodesmium colonies collected at two stations in the western tropical South Pacific. Sampling at a third station located in the oligotrophic South Pacific Gyre revealed no Trichodesmium but showed presence of UCYN-B, although no nifH expression was detected. Our results suggest that Trichodesmium behaves mixotrophically in response to certain environmental conditions, providing them with metabolic plasticity and adding up to the view that mixotrophy is widespread among marine microbes.

Toxicological impact of deltamethrin on growth and nitrogen content of a rice field cyanobacterium Calothrix sp. (GUEco 1002).

Cyanobacteria are an important component in the rice field ecosystem and are a well known source of natural biofertilizer. Pesticidal application for the control of pests in rice field soil has led to several environmental problems, and poses a great threat to these beneficial microorganisms. Studies on the impact of pesticides on the diazotrophic growth and survivability of these microorganisms have recently gained much attention. The present paper describes the effects of an iterated use of the insecticide deltamethrin (2.8% EC) on the growth and nitrogen fixation capacity of the filamentous cyanobacterium Calothrix sp. (strain GUEco 1002). This organism has shown a varying degree of sensitivity to the insecticide. For evaluating the deltamethrin toxicity, the test organism was subjected to varying concentrations of deltamethrin i.e. 17.5 ppm, 35 ppm, 70 ppm and 140 ppm based upon LC50 for 20 days. The data obtained in the laboratory revealed that the treatment of the test organism with deltamethrin (17.5-140 ppm) negatively affected its growth, pigments, protein and nitrogen content in a time dose dependent manner. In contrast, carbohydrate content significantly increased with increasing concentrations of deltamethrin, this effect being more prominent at 140 ppm treatment (38%). At this high level (140 ppm), the test organism showed a significant decrease in dry weight biomass (46%), chlorophyll-a (72%), carotenoids (57%), phycocyanin (67%), protein (69%) and nitrogen content (61%) over the control. A little, but insignificant, stimulatory effect on nitrogen content was recorded at 17.5 ppm of the insecticide which however, was the opposite in the case of growth, pigments, carbohydrate and protein content.

Phosphorus deficiency induced by aluminum in a marine nitrogen-fixing cyanobacterium Crocosphaera watsonii WH0003.

Large amounts of aluminum (Al) enter the ocean through atmospheric dust deposition and river runoffs. However, few studies have reported the effects of Al on marine phytoplankton, especially nitrogen-fixing cyanobacteria. By using the isotope tracer method and quantitative reverse transcription PCR (RT-qPCR), we examined the physiological effect of Al (0.2, 2 and 20 µM) on the unicellular marine nitrogen-fixing cyanobacterium Crocosphaera watsonii in Aquil* medium. We show that Al has an inhibitory physiological effect on C. watsonii, including changes in growth rate, nitrogen fixation rate, carbon fixation rate, cell size, fast rise chlorophyll fluorescence kinetics, cellular photosynthetic pigment and C/N/P content, the same as that of the phosphorus deficient treatment. The ratio of cellular elements C:N:P showed that phosphorus was deficient in the cell of C. watsonii after Al treatment (2 and 20 µM). In addition, Al stimulated the expression of phosphorus-related genes pstS, phoH, phoU, ppK and ppX in C. watsonii. All these results suggest that Al-treated C. watsonii is phosphorus-limited, and that the phosphorus deficiency induced by Al may be one mechanism behind aluminum's toxicity.

Succession of microbial populations and nitrogen-fixation associated with the biodegradation of sediment-oil-agglomerates buried in a Florida sandy beach.

The Deepwater Horizon (DWH) oil spill contaminated coastlines from Louisiana to Florida, burying oil up to 70 cm depth in sandy beaches, posing a potential threat to environmental and human health. The dry and nutrient-poor beach sand presents a taxing environment for microbial growth, raising the question how the biodegradation of the buried oil would proceed. Here we report the results of an in-situ experiment that (i) characterized the dominant microbial communities contained in sediment oil agglomerates (SOAs) of DWH oil buried in a North Florida sandy beach, (ii) elucidated the long-term succession of the microbial populations that developed in the SOAs, and (iii) revealed the coupling of SOA degradation to nitrogen fixation. Orders of magnitude higher bacterial abundances in SOAs compared to surrounding sands distinguished SOAs as hotspots of microbial growth. Blooms of bacterial taxa with a demonstrated potential for hydrocarbon degradation (Gammaproteobacteria, Alphaproteobacteria, Actinobacteria) developed in the SOAs, initiating a succession of microbial populations that mirrored the evolution of the petroleum hydrocarbons. Growth of nitrogen-fixing prokaryotes or diazotrophs (Rhizobiales and Frankiales), reflected in increased abundances of nitrogenase genes (nifH), catalyzed biodegradation of the nitrogen-poor petroleum hydrocarbons, emphasizing nitrogen fixation as a central mechanism facilitating the recovery of sandy beaches after oil contamination.

Interactive Effects of Nitrogen-Fixing Bacteria Inoculation and Nitrogen Fertilization on Soybean Yield in Unfavorable Edaphoclimatic Environments.

The objective of this work was to evaluate the effect of nitrogen (N) fertilization and the biological nitrogen fixation efficiency in soybean crops grown in unfavorable environments (high temperature, low fertility, and areas with sandy soil). Six field experiments were conducted between 2015 and 2018. Two experiments were performed per year. They were conducted in two separate areas. One was previously covered with degraded pasture (post-pasture area). The other was previously used to plant soybean (post-soybean crop area). The treatments consisted of inoculations with N-fixing bacteria (NFB) (0, 4, 8, and 12 doses ha-1) and N fertilization with rates of 0, 25, 50, and 100 kg ha-1. N fertilization and inoculation with NFB increased soil N, though the levels were still low. Among the tested groups, those with the application of eight doses of inoculant recorded the highest grain yields in post-soybean areas. They showed 10% (237 kg ha-1) and 15% (336 kg ha-1) higher grain yields when compared to crops treated without inoculant and crops with four doses of inoculant, respectively. N fertilization with 25 and 100 kg ha-1 decreased the root nodules of soybean plants grown in the post-soybean and post-pasture areas, respectively. Soybean crops grown on degraded pasture areas also showed good response to N fertilization (50 kg ha-1) when combined with NFB inoculation (12 doses ha-1). These showed grain yields 22% (439 kg ha-1) higher than those of plants treated with just 12 doses of inoculant and no N fertilization.

High heavy metal load does not inhibit nitrogen fixation in moss-cyanobacteria associations.

Nitrogen (N2) fixation by moss-associated cyanobacteria is one of the main sources of new N input in pristine ecosystems such as boreal forests and arctic tundra. Given the non-vascular physiology of mosses, they are especially sensitive to e.g. increased N input and heavy metal deposition. While the effects of increased N input on moss-associated N2 fixation has been comprehensively assessed, hardly any reports exist on the effects of increased heavy metal load on this key ecosystem function. To address this knowledge gap, we made use of an extreme metal pollution gradient in boreal forests of Northern Sweden originating from a metal mine and its associated smelters. We collected the common moss Pleurozium schreberi, known to host cyanobacteria, along a distance gradient away from the metal source of pollution and measured moss-metal content (Fe, Cu, Zn, Pb) as well as N2 fixation. We found a strong distance gradient in moss-metal content for all investigated metals: a sharp decline in metal content with distance away from the metal pollution source. However, we found a similarly steep gradient in moss-associated N2 fixation, with highest activity closest to the metal source of pollution. Hence, while mosses may be sensitive to increased heavy metal inputs, the activity of colonising cyanobacteria seem to be unaffected by heavy metals, and consequently, ecosystem function may not be compromised by elevated metal input.

Active nitrogen fixation by Crocosphaera expands their niche despite the presence of ammonium - A case study.

Unicellular nitrogen fixer Crocosphaera contributes substantially to nitrogen fixation in oligotrophic subtropical gyres. They fix nitrogen even when significant amounts of ammonium are available. This has been puzzling since fixing nitrogen is energetically inefficient compared with using available ammonium. Here we show that by fixing nitrogen, Crocosphaera can increase their population and expand their niche despite the presence of ammonium. We have developed a simple but mechanistic model of Crocosphaera based on their growth in steady state culture. The model shows that the growth of Crocosphaera can become nitrogen limited despite their capability to fix nitrogen. When they fix nitrogen, the population increases by up to 78% relative to the case without nitrogen fixation. When we simulate a simple ecological situation where Crocosphaera exists with non-nitrogen-fixing phytoplankton, the relative abundance of Crocosphaera increases with nitrogen fixation, while the population of non-nitrogen-fixing phytoplankton decreases since a larger fraction of fixed nitrogen is consumed by Crocosphaera. Our study quantitatively supports the benefit of nitrogen fixation despite the high electron/energy costs, even when an energetically efficient alternative is available. It demonstrates a competitive aspect of Crocosphaera, permitting them to be regionally significant nitrogen fixers.

Cyanobacteria as Nanogold Factories II: Chemical Reactivity and anti-Myocardial Infraction Properties of Customized Gold Nanoparticles Biosynthesized by Cyanothece sp.

Cyanothece sp., a coccoid, unicellular, nitrogen-fixing and hydrogen-producing cyanobacterium, has been used in this study to biosynthesize customized gold nanoparticles under certain chemical conditions. The produced gold nanoparticles had a characteristic absorption band at 525-535 nm. Two types of gold nanoparticle, the purple and blue, were formed according to the chemical environment in which the cyanobacterium was grown. Dynamic light scattering was implemented to estimate the size of the purple and blue nanoparticles, which ranged from 80 ± 30 nm and 129 ± 40 nm in diameter, respectively. The highest scattering of laser light was recorded for the blue gold nanoparticles, which was possibly due to their larger size and higher concentration. The appearance of anodic and cathodic peaks in cyclic voltammetric scans of the blue gold nanoparticles reflected the oxidation into gold oxide, followed by the subsequent reduction into the nano metal state. The two produced forms of gold nanoparticles were used to treat isoproterenol-induced myocardial infarction in experimental rats. Both forms of nanoparticles ameliorated myocardial infarction injury, with a slight difference in their curative activity with the purple being more effective. Mechanisms that might explain the curative effect of these nanoparticles on the myocardial infarction were proposed. The morphological, physiological, and biochemical attributes of the Cyanothece sp. cyanobacterium were fundamental for the successful production of "tailored" nanoparticles, and complemented the chemical conditions for the differential biosynthesis process. The present research represents a novel approach to manipulate cyanobacterial cells towards the production of different-sized gold nanoparticles whose curative impacts vary accordingly. This is the first report on that type of manipulated gold nanoparticles biosynthesis which will hopefully open doors for further investigations and biotechnological applications.

The Pseudomonas stutzeri-Specific Regulatory Noncoding RNA NfiS Targets katB mRNA Encoding a Catalase Essential for Optimal Oxidative Resistance and Nitrogenase Activity.

Pseudomonas stutzeri A1501 is a versatile nitrogen-fixing bacterium capable of living in diverse environments and coping with various oxidative stresses. NfiS, a regulatory noncoding RNA (ncRNA) involved in the control of nitrogen fixation in A1501, was previously shown to be required for optimal resistance to H2O2; however, the precise role of NfiS and the target genes involved in the oxidative stress response is entirely unknown. In this work, we systematically investigated the NfiS-based mechanisms underlying the response of this bacterium to H2O2 at the cellular and molecular levels. A mutant strain carrying a deletion of nfiS showed significant downregulation of oxidative stress response genes, especially katB, a catalase gene, and oxyR, an essential regulator for transcription of catalase genes. Secondary structure prediction revealed two binding sites in NfiS for katB mRNA. Complementation experiments using truncated nfiS genes showed that each of two sites is functional, but not sufficient, for NfiS-mediated regulation of oxidative stress resistance and nitrogenase activities. Microscale thermophoresis assays further indicated direct base pairing between katB mRNA and NfiS at both sites 1 and 2, thus enhancing the half-life of the transcript. We also demonstrated that katB expression is dependent on OxyR and that both OxyR and KatB are essential for optimal oxidative stress resistance and nitrogenase activities. H2O2 at low concentrations was detoxified by KatB, leaving O2 as a by-product to support nitrogen fixation under O2-insufficient conditions. Moreover, our data suggest that the direct interaction between NfiS and katB mRNA is a conserved and widespread mechanism among P. stutzeri strains.IMPORTANCE Protection against oxygen damage is crucial for survival of nitrogen-fixing bacteria due to the extreme oxygen sensitivity of nitrogenase. This work exemplifies how the small ncRNA NfiS coordinates oxidative stress response and nitrogen fixation via base pairing with katB mRNA and nifK mRNA. Hence, NfiS acts as a molecular link to coordinate the expression of genes involved in oxidative stress response and nitrogen fixation. Our study provides the first insight into the biological functions of NfiS in oxidative stress regulation and adds a new regulation level to the mechanisms that contribute to the oxygen protection of the MoFe nitrogenase.

Plant-based oil-sorbents harbor native microbial communities effective in spilled oil-bioremediation under nitrogen starvation and heavy metal-stresses.

Cultivation on selective media revealed that the oil-sorbents, wheat straw, corncobs and sugarcane bagasse harbor hydrocarbonoclastic, diazotrophic and heavy metal-resistant microorganisms. Nitrogen-free media containing 1.0% crude oil lost between 32.2 and 37.5% of this oil, after 8 months when they have been inoculated with such microorganism-loaded sorbents. The used wheat straw, corncobs and sugarcane bagasse samples, 1.0 g each, absorbed respectively, 1.9, 1.1 and 2.5 g oil samples, and lost 24.3-39.2% of these amounts, after they had been incubated for 8 months. Total genomic DNA's from culture media and sorbents revealed various nitrogenase-coding nifH-genes. Pure hydrocarbonoclastic microbial isolates tolerated certain concentrations of, Hg2+, Cd2+, Pb2+, AsO43- and AsO33-. Some of those isolates even grew excellently with up to 1000 ppm of Pb2+ and 36,000 ppm of AsO43- also in the presence of oil. Tested strains removed the tested heavy metals, Hg2+, Cd2+ and Pb2+ from the media and thus, reduced their toxicity against the hydrocarbon-degraders. It was concluded that plant-based sorbents, not only remove oil physically, but also harbor microbial communities effective in spilled oil-bioremediation under multiple stresses. Although each community consisted of one to three species only, the consortia which reached in numbers millions of CFU ml-1 enrich the oily media with fixed nitrogen, and remove heavy metals which otherwise inhibit the oil-degrading microorganisms.