Nitrate Increases Membrane Stability of Potato Cells under Anoxia

Summary Potato cells (Solanum tuberosum L.), cultivated in original Murashige-Skoog (MS) medium for 5 days were subsequently transferred into {MS} media containing nitrate or ammonium as sole inorganic N source and incubated under anoxia for 24 h. With regard to lipid stability, these cells behaved differently. Although lipid hydrolysis occurred in both cases by the same mechanism, nitrate was able to postpone free fatty acid release for about 6 h compared with ammonium within the 24 h anoxia treatment. The increased membrane lipid stability of nitrate-treated cells under anoxia was correlated with a higher nitrate reduction capability and an improved energy status.

Green synthesis of polypyrrole-supported metal catalysts: application to nitrate removal in water

Pt and Pt/Sn catalysts supported on polypyrrole (PPy) have been prepared using Ar plasma to reduce the metal precursors dispersed on the polymer. The PPy support was synthesized by chemical polymerization of pyrrole with FeCl3·6H2O, this leading to the conducting form of the polymer (conductimetric measurements). The Ar plasma treatment produced a partial reduction of platinum ions, anchored as platinum chloro-complexes to the PPy chain, into metallic platinum. A homogeneous distribution of Pt and Sn nanoparticles was observed by TEM. Activity of the PPy-supported catalysts was evaluated in the reduction of aqueous nitrate with H2 at room temperature. Nitrate concentration in water below the maximum acceptable level of 50 mg L−1 was achieved with all catalysts. However, considering not only efficiency in nitrate reduction, but also minimized concentrations of undesired nitrite and ammonium, the monometallic Pt catalyst seems to be the most promising one.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

Role of degree of saturation in denitrification of unsaturated sand specimens

Nitrate contamination of groundwater arises from anthropogenic activities, such as, fertilizer and animal manure applications and infiltration of wastewater/leachates. During migration of wastewater and leachates, the vadose zone (zone residing above the groundwater table), is considered to facilitate microbial denitrification. Particle voids in vadose zone are deficient in dissolved oxygen as the voids are partially filled by water and the remainder by air. Discontinuities in liquid phase would also restrict oxygen diffusion and therefore facilitate denitrification in the vadose/unsaturated soil zone. The degree of saturation of soil specimen (S (r)) quantifies the relative volume of voids filled with air and water. Unsaturated specimens have S (r) values ranging between 0 and 100 %. Earlier studies from naturally occurring nitrate losses in groundwater aquifers in Mulbagal town, Kolar District, Karnataka, showed that the sub-surface soils composed of residually derived sandy soil; hence, natural sand was chosen in the laboratory denitrification experiments. With a view to understand the role of vadose zone in denitrification process, experiments are performed with unsaturated sand specimens (S (r) = 73-90 %) whose pore water was spiked with nitrate and ethanol solutions. Experimental results revealed 73 % S (r) specimen facilitates nitrate reduction to 45 mg/L in relatively short durations of 5.5-7.5 h using the available natural organic matter (0.41 % on mass basis of sand); consequently, ethanol addition did not impact rate of denitrification. However, at higher S (r) values of 81 and 90 %, extraneous ethanol addition (C/N = 0.5-3) was needed to accelerate the denitrification rates.

Nitrogen dynamics in large shallow eutrophic Lake Chaohu, China

Temporal and spatial dynamics of nitrogen in lake and interstitial water were studied monthly in a large shallow, eutrophic lake in subtropical China from October 2002 to September 2003. The distribution of nitrogen was consistent with the idea that high nitrogen concentrations in the western part of the lake resulted from high levels of the nutrients from the surrounding cities through sewage-drainage systems. Nitrate was the predominant form of nitrogen in the overlying water, while ammonium was predominant in the interstitial water, indicating that strong oxidative nutrient regeneration occurred near the sediment-water interface. Nitrate could be an important dissolved inorganic matter source for phytoplankton, which in turn influenced the seasonal variations of nitrate concentrations in lake water. Significant positive correlation between ammonium fluxes and water temperature was observed and could probably be attributed to the intensified ammonification and nitrate reduction with increased temperature. Positive correlation between ammonium fluxes and algae biomass and Chl a concentrations may indicate that phytoplankton was an important factor driving ammonium fluxes in our study lake, and vice versa that higher fluxes of ammonium supported a higher biomass of the phytoplankton.

化感物质香豆素对苜蓿种子萌发及幼苗初级氮同化的影响

香豆素类物质是苯丙酸内酯（环酯）类化合物，绝大部分高等植物通过次生代谢途径都能合成。研究表明，香豆素类物质是花椒体内最重要的化感物质，系统研究香豆素类物质的作用机理有助于理解和最终解决花椒连作障碍。本文通过研究香豆素对几种植物种子特别是苜蓿种子萌发、苜蓿幼苗初级氮同化的影响，从生理生化角度揭示香豆素的作用方式，为花椒连作障碍的解决和化感作用机制的深入理解提供依据。主要研究结果如下：1. 研究了香豆素对6 种常见作物种子萌发的影响，并对一组数据采用4个不同的指标进行评价，对生物测定化感作用中存在的问题进行了讨论。结果发现1.0mM的香豆素对采用的6 种作物的种子萌发均表现出一定的化感作用，4 个指标的敏感程度依次为S (发芽速度)＞AS(累积发芽速度)＞CRG(发芽指数)＞GT(最终发芽率)。种子萌发实验是化感作用研究中最重要、应用最广泛的生物测定方法之一，应根据不同的研究目的合理采用指标和实验方法。2. 采用培养皿试纸法进行种子萌发试验，研究了香豆素水溶液在苜蓿种子萌发过程中对其吸水、电导率及抗氧化保护酶活性的影响。结果表明，影响苜蓿种子发芽的香豆素浓度阀值为0.3mM。香豆素在1.0mM 的浓度下降低了苜蓿种子吸水阶段Ⅱ的吸水速度，使其外渗物质增多，电导率增大，并显著抑制了超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)的活性，同时种子体内丙二醛（MDA）的含量显著增大。高浓度香豆素破坏了膜的结构、影响了抗氧化保护酶的活性是香豆素降低苜蓿发芽率的原因之一，也可能是影响花椒-苜蓿间作的关键因素之一。3. 不同浓度（0、25 μM、50 μM、0.1 mM、1.0 mM）化感活性物质香豆素对10 日龄苜蓿幼苗初级氮同化的影响的结果表明25 µM～50 µM 的香豆素加快了苜蓿幼苗对硝态氮的吸收。高浓度的香豆素导致苜蓿根系和叶片内可溶性蛋白含量降低、鲜重减小、地下鲜重/地上鲜重（R/S）的比值升高，根系中初级氮同化的关键酶硝酸还原酶（NR）、谷氨酸胺合成酶（GS）、谷氨酸脱氢酶（GDH）的活性降低，叶片中NR、GS 的活性减低、叶绿素含量减少，而GDH 的活性升高。香豆素影响苜蓿幼苗氮代谢和氨同化的关键酶，导致体内养分的缺失是香豆素抑制苜蓿幼苗生长的机理之一。Coumarins are lactones of o-hydroxycinnamic acid, and are allelopathiccompounds that originate in the phenylpropanoid pathway. They are synthesized byalmost all higher plants. According to previous studies, coumarins were mostimportant allelochemicals in Chinese prickly ash. Systematically research of theeffect of coumarin could help to comprehend the continuous cropping impediment.The effects of coumarin on seed germination and primary nitrogen assimilation ofalfalfa were studied. The main results showed that:1. We compared four common germination indices (S, AS, CRG, GT)preciously calculated with the same date. The results showed that, at theconcentration of 1.0 mM, coumarin inhibited seeds germination. Among all indices,the S index was most sensitive, followed by the AS and CRG indices. Andsuggestions on the expression of bioassay results were also provided.2. At concentrations above 0.3 mM, coumarin inhibited seed germination in aconcentration-dependent manner. During seed imbibitionⅡ, coumarin at 1.0 mMsignificantly reduced the activities of superoxide dismutase (SOD), catalase (CAT),peroxidase (POD), while the content of malonyldialdehyde (MDA) in alfalfa seedssignificantly increased. The higher concentration coumarin destroyed structure ofmembrane and influenced activities of antioxidant enzymes, which might be one ofthe reasons that coumarin decreased germination rate of alfalfa, and one of the keyfactors influencing Chinese prickly ash-alfalfa intercropping.3. Alfalfa plants were exposed to different concentration of coumarin (0、25μM、50 μM、0.1 mM、1.0 mM) grown for 10 days on control medium. Coumarin, in the range of 25 μM～50 μM, significantly stimulated the net nitrate uptake.Increasing coumarin concentration led to a decrease of protein contents in theleaves and roots. The root to shoot (R/S) FW ratio was increased by increasingcoumarin concentration. Under high coumarin concentration, the activities of nitratereductase (NR) and glutamine synthetase (GS) were repressed in the roots andleaves. Glutamate dehydrogenase (GDH) was inhibited in the roots, while enhancedin the leaves. Chlorophyll contents in the leaves were also decreased under highcoumain concentration. Coumarin decreased alfalfa growth by (i) nutritionaldeficiencies shown by the decrease of nitrate, (ii) lowered N compound synthesisvia inhibition of nitrate reduction and ammonium assimilation.

Descarga costeira de águas subterrâneas com elevado conteúdo em nitratos para a Ria Formosa

Dissertação de Mestrado, Estudos Marinhos e Costeiros, Faculdade de Ciências do Mar e Ambiente, Universidade do Algarve, 2007

Nitrogen Metabolism in leaves of a tank epiphytic bromeliad: Characterization of a spatial and functional division

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO(3)(-)/NH(4)(+) or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary. GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH(4)(+) assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad`s leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo de bactérias do gênero Gluconobacter: isolamento, purificação, identificação fenotípica e molecular

A importância do estudo de bactérias acéticas, em especial as do gênero Gluconobacter, está baseada em suas aplicações industriais, pois estas possuem a capacidade de bioconversão de sorbitol a sorbose, viabilizando o processo de produção de vitamina C. O estudo envolveu coletas de amostras em indústrias de refrigerante, flores, frutos e mel, seguidas de purificação, identificação fenotípica e identificação molecular, com a utilização de iniciador definido a partir de consulta ao Nucleotide Sequence Database. Preservaram-se as linhagens identificadas como membros da família Acetobacteriaceae, gênero Gluconobacter. Foi isolado um total de 110 linhagens dos substratos: Pyrostegia venusta (Cipó de São João), mel, Vitis vinifera (uva), Pyrus communis (pêra), Malus sp. (maçã) e de duas amostras de refrigerantes envasados em embalagens de PET de 2 L. Deste total, 57 linhagens foram recuperadas em meio MYP (manitol, extrato de levedura, peptona), 12 em meio YGM (glicose, manitol, extrato de levedura, etanol, ácido acético), 41 em meio de enriquecimento e, posteriormente, em meio GYC (glicose, extrato de levedura e carbonato de cálcio). Obtiveram-se 68 linhagens identificadas como bastonetes Gram negativos. Destas, 31 foram caracterizadas bioquimicamente como pertencentes à família Acetobacteriaceae por serem catalase positivas, oxidase negativas e produtoras de ácido a partir de glicose. A caracterização dessas linhagens foi complementada com os testes bioquímicos: liquefação da gelatina, redução de nitrato, formação de indol e H2S e oxidação de etanol a ácido acético. Métodos moleculares foram aplicados para identificação do gênero Gluconobacter. Finalmente, oito linhagens foram caracterizadas como pertencentes ao gênero Gluconobacter. As linhagens encontram-se depositadas em coleção de cultura do laboratório de Microbiologia do Departamento de Biologia da UNESP, campus de Assis, estocadas em extrato de malte 20 a -196 ºC.

Evaluation of the microbial diversity of denitrifying bacteria in batch reactor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do nitrogênio e da combinação das formas de nitrogênio na atividade das enzimas redutase do nitrato e glutamina sintetase em milho (Zea mays L.) e na produtividade

Pós-graduação em Agronomia - FEIS

Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes

A taxonomic and annotated functional description of microbial life was deduced from 53 Mb of metagenomic sequence retrieved from a planktonic fraction of the Neotropical high Andean (3,973 meters above sea level) acidic hot spring El Coquito (EC). A classification of unassembled metagenomic reads using different databases showed a high proportion of Gammaproteobacteria and Alphaproteobacteria (in total read affiliation), and through taxonomic affiliation of 16S rRNA gene fragments we observed the presence of Proteobacteria, micro-algae chloroplast and Firmicutes. Reads mapped against the genomes Acidiphilium cryptum JF-5, Legionella pneumophila str. Corby and Acidithiobacillus caldus revealed the presence of transposase-like sequences, potentially involved in horizontal gene transfer. Functional annotation and hierarchical comparison with different datasets obtained by pyrosequencing in different ecosystems showed that the microbial community also contained extensive DNA repair systems, possibly to cope with ultraviolet radiation at such high altitudes. Analysis of genes involved in the nitrogen cycle indicated the presence of dissimilatory nitrate reduction to N2 (narGHI, nirS, norBCDQ and nosZ), associated with Proteobacteria-like sequences. Genes involved in the sulfur cycle (cysDN, cysNC and aprA) indicated adenylsulfate and sulfite production that were affiliated to several bacterial species. In summary, metagenomic sequence data provided insight regarding the structure and possible functions of this hot spring microbial community, describing some groups potentially involved in the nitrogen and sulfur cycling in this environment. Citation: Jimenez DJ, Andreote FD, Chaves D, Montana JS, Osorio-Forero C, et al. (2012) Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes. PLoS ONE 7(12): e52069. doi:10.1371/journal.pone.0052069

Das GAF-Domänen-Protein NreA : ein neuartiger Nitratrezeptor aus Staphylococcus carnosus

Staphylococcus carnosus ist ein fakultativ anaerobes Bakterium, das aerobe Atmung, anaerobe Nitratatmung und Gärungsstoffwechsel betreiben kann. Die Expression des Nitratstoffwechsels wird durch das Dreikomponentensystem NreABC reguliert.rnUnter anaeroben Bedingungen besitzt die Sensorhistidinkinase NreB in ihrer PAS-Domäne ein [Fe4S4]2+-Cluster. Das aktive (anaerobe) [Fe4S4]2+-NreB überträgt nach Autophosphorylierung die Phosphorylgruppe auf den Antwortregulator NreC, welcher dann die Expression der Gene der Nitratatmung aktiviert. Nitrat wirkt mit Hilfe des NreA-Proteins auf diese Gene induzierend. Im Rahmen der vorliegenden Arbeit wurde gezeigt, dass NreA ein GAF-Domänen-Protein und ein neuartiger Nitratrezeptor ist.rnDie Natur von NreA als GAF-Domänen-Protein bestätigte sich beim Vergleich der Kristallstruktur mit denen anderer GAF-Domänen. GAF-Domänen sind weit verbreitet und binden typischer Weise kleine Moleküle. Als physiologischer Ligand von NreA zeigte sich Nitrat, das innerhalb einer definierten Bindetasche gebunden wird. NreA bindet vermutlich in dimerer Form an dimeres NreB und inhibiert dadurch die Phosphorylierung der Sensorhistidinkinase NreB. Die Interaktion von NreA mit NreB wurde in vivo durch BACTH-Messungen und sowohl in vivo als auch in vitro durch Cross-Linking Experimente gezeigt. Nitrat reduziert den Ergebnissen nach die Interaktion von NreA mit NreB.rnDurch Sequenzvergleiche von NreA mit Homologen wurden konservierte Aminosäuren identifiziert. Über gerichtete Mutagenese wurden 25 NreA-Varianten hergestellt und bezüglich ihres Verhaltens in Abhängigkeit von Nitrat in narG-lip-Reportergenstudien getestet. Anhand ihres Phänotyps wurden sie als Wildtyp, NreA- und NreABC-Mutanten klassifiziert. Die Nitratbindetasche war in sechs Fällen betroffen. Die Phänotypen der Mutationen in der Peripherie lassen sich mit Auswirkungen auf die vermutete Konformationsänderung oder auf die Interaktion mit NreB erklären. Mutationen von konservierten, oberflächenexponierten Resten führten vermehrt zu NreA/ON-Varianten. Es ließen sich Bereiche auf der Proteinoberfläche identifizieren, die für NreA/NreA- oder NreA/NreB-Interaktionen wichtig sein könnten.rnDie Untersuchungen zeigten, dass NreA mit NreB interagiert und dass dadurch ein NreA/NreB-Sensorkomplex für die gemeinsame Erkennung von Nitrat und Sauerstoff gebildet wird.

Extreme climatic events: impacts of drought and high temperature on physiological processes in agronomically important plants

Climate models predict more frequent and more severe extreme events (e.g., heat waves, extended drought periods, flooding) in many regions for the next decades. The impact of adverse environmental conditions on crop plants is ecologically and economically relevant. This review is focused on drought and heat effects on physiological status and productivity of agronomically important plants. Stomatal opening represents an important regulatory mechanism during drought and heat stress since it influences simultaneously water loss via transpiration and CO2 diffusion into the leaf apoplast which further is utilized in photosynthesis. Along with the reversible short-term control of stomatal opening, stomata and leaf epidermis may produce waxy deposits and irreversibly down-regulate the stomatal conductance and non-stomatal transpiration. As a consequence photosynthesis will be negatively affected. Rubisco activase—a key enzyme in keeping the Calvin cycle functional—is heat-sensitive and may become a limiting factor at elevated temperature. The accumulated reactive oxygen species (ROS) during stress represent an additional challenge under unfavorable conditions. Drought and heat cause accumulation of free amino acids which are partially converted into compatible solutes such as proline. This is accompanied by lower rates of both nitrate reduction and de novo amino acid biosynthesis. Protective proteins (e.g., dehydrins, chaperones, antioxidant enzymes or the key enzyme for proline biosynthesis) play an important role in leaves and may be present at higher levels under water deprivation or high temperatures. On the whole plant level, effects on long-distance translocation of solutes via xylem and phloem and on leaf senescence (e.g., anticipated, accelerated or delayed senescence) are important. The factors mentioned above are relevant for the overall performance of crops under drought and heat and must be considered for genotype selection and breeding programs.