Atributos químicos, bioquímicos e microbiológicos em solos com 18 anos de aplicações anuais de lodo de esgoto

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The NreABC system of Staphylococcus carnosus combines nitrate and oxygen sensing by an NreA,NreB sensor complex

Staphylococcus carnosus is a facultative anaerobic bacterium which features the cytoplasmic NreABC system. It is necessary for regulation of nitrate respiration and the nitrate reductase gene narG in response to oxygen and nitrate availability. NreB is a sensor kinase of a two-component system and represents the oxygen sensor of the system. It binds an oxygen labile [4Fe-4S]2+ cluster under anaerobic conditions. NreB autophosphorylates and phosphoryl transfer activates the response regulator NreC which induces narG expression. The third component of the Nre system is the nitrate receptor NreA. In this study the role of the nitrate receptor protein NreA in nitrate regulation and its functional and physiological effect on oxygen regulation and interaction with the NreBC two-component system were detected. In vivo, a reporter gene assay for measuring expression of the NreABC regulated nitrate reductase gene narG was used for quantitative evaluation of NreA function. Maximal narG expression in wild type S. carnosus required anaerobic conditions and the presence of nitrate. Deletion of nreA allowed expression of narG under aerobic conditions, and under anaerobic conditions nitrate was no longer required for maximal induction. This indicates that NreA is a nitrate regulated inhibitor of narG expression. Purified NreA and variant NreA(Y95A) inhibited the autophosphorylation of anaerobic NreB in part and completely, respectively. Neither NreA nor NreA(Y95A) stimulated dephosphorylation of NreB-phosphate, however. Inhibition of phosphorylation was relieved completely when NreA with bound nitrate (NreA•[NO3-]) was used. The same effects of NreA were monitored with aerobically isolated Fe-S-less NreB, which indicates that NreA does not have an influence on the iron-sulfur cluster of NreB. In summary, the data of this study show that NreA interacts with the oxygen sensor NreB and controls its phosphorylation level in a nitrate dependent manner. This modulation of NreB-function by NreA and nitrate results in nitrate/oxygen co-sensing by an NreA/NreB sensory unit. It transmits the regulatory signal from oxygen and nitrate in a joint signal to target promoters. Therefore, nitrate and oxygen regulation of nitrate dissimilation follows a new mode of regulation not present in other facultative anaerobic bacteria.

Sulfate and Nitrate Assimilation in Leaves of Quercus ilex and Quercus pubescens Grown Near Natural CO2 Springs in Central Italy

The effect of long-term exposure to elevated pCO2 concentrations on sulfate and nitrate assimilation was studied under field conditions using leaves from Quercus ilex and Quercus pubescens trees growing with ambient or elevated CO2 concentrations in the vicinity of three natural CO2 springs, Bossoleto, Laiatico and Sulfatara, in Tuscany, Italy. The activity of the key enzymes of sulfate assimilation, adenosine 5′-phosphosulfate reductase (APR) and nitrate assimilation, nitrate reductase (NR), were measured together with the levels of acid soluble thiols, and soluble non-proteinogenic nitrogen compounds. Whereas NR activity remained unaffected in Q. ilex or increased Q. pubescence, APR activity decreased in the area of CO2 springs. The latter changes were often accompanied by increased GSH concentrations, apparently synthesized from H2S and SO2 present in the gas mixture emitted from the CO2 springs. Thus, the diminished APR activity in leaves of Q. ilex and Q. pubescence from spring areas can best be explained by the exposure to gaseous sulfur compounds. Although the concentrations of H2S and SO2 in the gas mixture emitted from the vents at the CO2 springs were low at the Bossoleto and Laiatico spring, these sulfur gases pose physiological effects, which may override consequences of elevated pCO2.

Dual pathways for regulation of root branching by nitrate

Root development is extremely sensitive to variations in nutrient supply, but the mechanisms are poorly understood. We have investigated the processes by which nitrate (NO3−), depending on its availability and distribution, can have both positive and negative effects on the development and growth of lateral roots. When Arabidopsis roots were exposed to a locally concentrated supply of NO3− there was no increase in lateral root numbers within the NO3−-rich zone, but there was a localized 2-fold increase in the mean rate of lateral root elongation, which was attributable to a corresponding increase in the rate of cell production in the lateral root meristem. Localized applications of other N sources did not stimulate lateral root elongation, consistent with previous evidence that the NO3− ion is acting as a signal rather than a nutrient. The axr4 auxin-resistant mutant was insensitive to the stimulatory effect of NO3−, suggesting an overlap between the NO3− and auxin response pathways. High rates of NO3− supply to the roots had a systemic inhibitory effect on lateral root development that acted specifically at the stage when the laterals had just emerged from the primary root, apparently delaying final activation of the lateral root meristem. A nitrate reductase-deficient mutant showed increased sensitivity to this systemic inhibitory effect, suggesting that tissue NO3− levels may play a role in generating the inhibitory signal. We present a model in which root branching is modulated by opposing signals from the plant’s internal N status and the external supply of NO3−.

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80–90°C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded β/α-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature1

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m−2 s−1) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m−2 s−1) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.

Wheat leaves emit nitrous oxide during nitrate assimilation

Nitrous oxide (N2O) is a key atmospheric greenhouse gas that contributes to global climatic change through radiative warming and depletion of stratospheric ozone. In this report, N2O flux was monitored simultaneously with photosynthetic CO2 and O2 exchanges from intact canopies of 12 wheat seedlings. The rates of N2O-N emitted ranged from <2 pmol⋅m−2⋅s−1 when NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} was the N source, to 25.6 ± 1.7 pmol⋅m−2⋅s−1 (mean ± SE, n = 13) when the N source was shifted to NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}. Such fluxes are among the smallest reported for any trace gas emitted by a higher plant. Leaf N2O emissions were correlated with leaf nitrate assimilation activity, as measured by using the assimilation quotient, the ratio of CO2 assimilated to O2 evolved. 15N isotopic signatures on N2O emitted from leaves supported direct N2O production by plant NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} assimilation and not N2O produced by microorganisms on root surfaces and emitted in the transpiration stream. In vitro production of N2O by both intact chloroplasts and nitrite reductase, but not by nitrate reductase, indicated that N2O produced by leaves occurred during photoassimilation of NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} in the chloroplast. Given the large quantities of NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} assimilated by plants in the terrestrial biosphere, these observations suggest that formation of N2O during NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} photoassimilation could be an important global biogenic N2O source.

Effects of nitrogen sources on the nitrate assimilation in Haloferax mediterranei: growth kinetics and transcriptomic analysis

The haloarchaeon Haloferax mediterranei is able to grow in a defined culture media not only in the presence of inorganic nitrogen salt but also with amino acid as the sole nitrogen source. Assimilatory nitrate and nitrite reductases, respectively, catalyze the first and second reactions. The genes involved in this process are nasA, which encodes nitrate reductase and is found within the operon nasABC, and nasD, which encodes nitrite reductase. These genes are subjected to transcriptional regulation, being repressed in the presence of ammonium and induced with either nitrate or nitrite. This type of regulation has also been described when the amino acids are used as nitrogen source in the minimal media. Furthermore, it has been observed that the microorganism growth depends on nitrogen source, obtaining the lowest growth rate in the presence of nitrate and aspartate. In this paper, we present the results of a comparative study of microorganism growth and transcriptomic analysis of the operon nasABC and gene nasD in different nitrogen sources. The results are the first ever produced in relation to amino acids as nitrogen sources within the Halobacteriaceae family.

Structure of the active site of sulfite dehydrogenase from Starkeya novella

In this paper, we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized and reduced active sites of the sulfite dehydrogenase from Starkeya novella. Our results provide the first direct structural information on the active site of the oxidized form of this enzyme and confirm the conclusions derived from protein crystallography that the molybdenum coordination is analogous to that of the sulfite oxidases. The molybdenum atom of the oxidized enzyme is bound by two Mo=O ligands at 1.73 angstrom and three thiolate Mo-S ligands at 2.42 angstrom, whereas the reduced enzyme has one oxo at 1.74 angstrom, one long oxygen at 2.19 angstrom (characteristic of Mo-OH2), and three Mo-S ligands at 2.40 angstrom.

Direct electrochemistry of human and rat NADPH cytochrome P450 reductase

The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cytochrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefully orchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function, no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purified human CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltammetry. For human CPR redox responses at -2 and -278 mV (with a ratio of 1e(-):3e(-)) vs NHE were seen at pH 7.9 while the potentials for rat CPR at pH 8.0 were -20 and -254 mV. All redox responses exhibit a pH dependence of approximately -59 mV/pH unit consistent with proton coupled electron transfer reactions of equal stoichiometry. (c) 2006 Elsevier B.V. All rights reserved.

The effect of nitrate and phosphate availability on Emiliania huxleyi(NZEH) physiology under different CO2 scenarios

Growth and calcification of the marine coccolithophorid Emiliania huxleyi is affected by ocean acidification and macronutrients limitation and its response varies between strains. Here we investigated the physiological performance of a highly calcified E. huxleyi strain, NZEH, in a multiparametric experiment. Cells were exposed to different CO2 levels (ranging from 250 to 1314 µatm) under three nutrient conditions [nutrient replete (R), nitrate limited (-N), and phosphate limited (-P)]. We focused on calcite and organic carbon quotas and on nitrate and phosphate utilization by analyzing the activity of nitrate reductase (NRase) and alkaline phosphatase (APase), respectively. Particulate inorganic (PIC) and organic (POC) carbon quotas increased with increasing CO2 under R conditions but a different pattern was observed under nutrient limitation. The PIC:POC ratio decreased with increasing CO2 in nutrient limited cultures. Coccolith length increased with CO2 under all nutrient conditions but the coccosphere volume varied depending on the nutrient treatment. Maximum APase activity was found at 561 ?atm of CO2 (pH 7.92) in -P cultures and in R conditions, NRase activity increased linearly with CO2. These results suggest that E. huxleyi's competitive ability for nutrient uptake might be altered in future high-CO2 oceans. The combined dataset will be useful in model parameterizations of the carbon cycle and ocean acidification.

Ensaio in vitro da enzima nitrato redutase e efeito da disponibilidade de nitrato e fosfato em variantes pigmentares de Hypnea musciformis (Wulfen) J. V. Lamour. (Gigartinales, Rhodophyta)

A enzima nitrato redutase (NR) catalisa a redução do nitrato a nitrito e controla a taxa de assimilação do nitrato. O ensaio in vitro da nitrato redutase foi otimizado para a linhagem selvagem (marrom, MA) e para a linhagem deficiente em ficoeritrina (verde-clara, VC) de Hypnea musciformis. As duas linhagens foram cultivadas em temperatura de 23 ± 2°C, fotoperíodo de 14 horas, irradiância de 60-90µmol fótons m-2s-1, e meio composto por água do mar esterilizada (30ups) enriquecida com a solução de von Stosch na concentração de 50% (VSES/2). As condições ótimas de ensaio para ambas as linhagens foram: 40µM de NADH; 10min de incubação do extrato bruto (EB) e 100µL de EB. A atividade ótima da NR ocorreu em 4 e 2mM de nitrato para a linhagem VC e MA, respectivamente. As linhagens VC e MA apresentaram, respectivamente, constante aparente de Michaelis-Menten (K M) para NADH de 0,2068 e 0,0837 µM, e K M para nitrato de 0,0492 e 0,0294mM. Os resultados indicam que a NR da linhagem MA tem maior afinidade pelo substrato do que a NR da linhagem VC de H. musciformis. Os experimentos para avaliar os efeitos da disponibilidade de nitrato (5 a 105µM) e nitrato e fosfato (0,5 a 25,5µM, com a relação N:P de 4:1) mostraram que a atividade da NR das linhagens VC e MA não aumentou com a adição de nitrato no meio, o que pode estar relacionado com o estado nutricional dessas algas. A atividade da NR foi maior nos tratamentos com adição de fosfato do que naqueles com adição de apenas nitrato, indicando que esse nutriente é importante para os processos metabólicos relacionados a atividade da NR.

Sugarcane genes associated with sucrose content

Background -: Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. Results -: We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. Conclusion -: Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.

Functional specialization of Eucalyptus fine roots: contrasting potential uptake rates for nitrogen, potassium and calcium tracers at varying soil depths

1. Little is known about the role of deep roots in the nutrition of forest trees and their ability to provide a safety-net service taking up nutrients leached from the topsoil. 2. To address this issue, we studied the potential uptake of N, K and Ca by Eucalyptus grandis trees (6 years of age - 25 m mean height), in Brazil, as a function of soil depth, texture and water content. We injected NO(3)(-)- (15)N, Rb(+) (analogue of K(+)) and Sr(2+) (analogue of Ca(2+)) tracers simultaneously in a solution through plastic tubes at 10, 50, 150 and 300 cm in depth in a sandy and a clayey Ferralsol soil. A complete randomized design was set up with three replicates of paired trees per injection depth and soil type. Recently expanded leaves were sampled at various times after tracer injection in the summer, and the experiment was repeated in the winter. Soil water contents were continuously monitored at the different depths in the two soils. 3. Determination of foliar Rb and Sr concentrations and (15)N atom % made it possible to estimate the relative uptake potential (RUP) of tracer injections from the four soil depths and the specific RUP (SRUP), defined as RUP, per unit of fine root length density in the corresponding soil layer. 4. The highest tracer uptake rates were found in the topsoil, but contrasting RUP distributions were observed for the three tracers. Whilst the RUP was higher for NO(3)(-)- (15)N than for Rb(+) and Sr(2+) in the upper 50 cm of soil, the highest SRUP values for Sr(2+) and Rb(+) were found at a depth of 300 cm in the sandy soil, as well as in the clayey soil when gravitational solutions reached that depth. 5. Our results suggest that the fine roots of E. grandis trees exhibit contrasting potential uptake rates with depth depending on the nutrient. This functional specialization of roots might contribute to the high growth rates of E. grandis trees, efficiently providing the large amounts of nutrients required throughout the development of these fast-growing plantations.

Effects of Molybdenum, Nickel, and Nitrogen Sources on the Mineral Nutrition and Growth of Rice Plants

Upland rice plants, cultivar `IAC 202,` were grown in nutrient solution until full tillering. Treatments consisted of ammonium nitrate (AN) or urea (UR) as nitrogen (N) source plus molybdenum (Mo) and/or nickel (Ni): AN + Mo + Ni, AN + Mo - Ni, AN - Mo + Ni, UR + Mo + Ni, UR + Mo - Ni, and UR - Mo + Ni. The experiment was carried out to better understand the effect of these treatments on dry-matter yield, chlorophyll, net photosynthesis rate, nitrate (NO3 --N), total N, in vitro activities of urease and nitrate reductase (NR), and Mo and Ni concentrations. In UR-grown plants, Mo and Ni addition increased yield of dry matter. Regardless of the N source, chlorophyll concentration and net photosynthesis rate were reduced when Mo or Ni were omitted, although not always significantly. The omission of either Mo or Ni led to a decrease in urease activity, independent of N source. Nitrate reductase activity increased in nutrient solutions without Mo, although NO3 --N increased. There was not a consistent variation in total N concentration. Molybdenum and Ni concentration in roots and shoots were influenced by their supply in the nutrient solution. Molybdenum concentration was not influenced by N sources, whereas Ni content in both root and shoots was greater in ammonium nitrate-grown plants. In conclusion, it can be hypothesized that there is a relationship between Mo and Ni acting on photosynthesis, although is an indirect one. This is the first evidence for a beneficial effect of Mo and Ni interaction on plant growth.