Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.     

Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula

It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.     

Comparative Proteomic Analysis of Soybean Nodulation Using a Supernodulation Mutant,SS2-2

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.     

Comparative proteomic analysis of soybean nodulation using a supernodulation mutant, SS2-2

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.     

Physiological Processes Contributing to the Difference in Grain Amino Acid Content between Two Hybrid Rice (Oryza sativa L.) Cultivars

Improving grain amino acid content of rice (Oryza sativa L.) is essential for the health of consumers. This study was conducted to identify the physiological processes that contribute to the higher grain amino acid content in hybrid rice cultivar Lingliangyou 268 compared to Luliangyou 996. The results showed that total amino acid content in grains was 9% higher in Lingliangyou 268 than in Luliangyou 996. There was no significant difference in grain nitrogen (N) content between Lingliangyou 268 and Luliangyou 996, while ratio of amino acid to N was 6% higher in Lingliangyou 268 compared to Luliangyou 996. A total of 16 differentially expressed proteins related to amino acid metabolism (e.g., erythronate-4-phosphate dehydrogenase domain containing protein) were identified in grains between Lingliangyou 268 and Luliangyou 996. The identified proteins were involved in 10 molecular functions. Six of the 10 defined functions were related to binding (heterocyclic compound binding, nucleoside phosphate binding, nucleotide binding, organic cyclic compound binding, protein binding, and small molecule binding) and the other 4 defined functions were catalytic activity, enzyme regulator activity, hydrolase activity, and transferase activity. These results indicate that the higher grain amino acid content in Lingliangyou 268 compared to Luliangyou 996 is attributed to increased efficiency of converting N to amino acid that results from altered expression of proteins related to amino acid metabolism.     

Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.     

Heterotrophic metabolism and diazotrophic growth ofNostoc sp. fromCycas circinalis

The cyanobiont ofCycas circinalis (identified asNostoc sp.) was isolated and its heterotrophic metabolism was studied in free culture under nitrogen-fixing conditions. Morphology, growth rate, nitrogenase activity, biochemical composition, efficiency of assimilation of organic carbon and molecular nitrogen were determined under different conditions of energy and carbon supply. The study has revealed the high potential of the heterotrophic metabolism in this symbiotic cyanobacterium. Although low rates of metabolic activities were attained under heterotrophic conditions, the efficiencies of organic carbon utilization (0.48 g cell-carbon per g glucose-carbon in chemoheterotrophy, from 0.65 to 0.74 under photoheterotrophy) and of N₂ assimilation (35.0 mg N₂ fixed per g glucose used in chemoheterotrophy, from 58.3 to 61.9 under photoheterotrophy) displayed by this organism were among the highest ever found in diazotrophically grown microorganisms. The isolate fromC. circinalis was able to grow indefinitely in the dark under nitrogen-fixing conditions, maintaining a well balanced biosynthetic activity and the capacity to resume photosynthetic metabolism quickly. The significance of the heterotrophic potential of this symbioticNostoc is discussed.     

NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation

Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.     

The exo-proteome and exo-metabolome of Nostoc punctiforme (Cyanobacteria) in the presence and absence of nitrate

The ability of nitrogen-fixing filamentous Cyanobacteria to adapt to multiple environments comes in part from assessing and responding to external stimuli, an event that is initiated in the extracellular milieu. While it is known that these organisms produce numerous extracellular substances, little work has been done to characterize both the metabolites and proteins present under standard laboratory growth conditions. We have assessed the extracellular milieu of Nostoc punctiforme when grown in liquid culture in the presence and absence of a nitrogen source (nitrate). The extracellular proteins identified were enriched in integrin β-propellor domains and calcium-binding sites with sequences unique to N. punctiforme, supporting a role for extracellular proteins in modulating species-specific recognition and behavior processes. Extracellular proteases are present and active under both conditions, with the cells grown with nitrate having a higher activity when normalized to chlorophyll levels. The released metabolites are enriched in peptidoglycan-derived tetrasaccharides, with higher levels in nitrate-free media.     

Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission

The amino acid glutamine has a central role in nitrogen metabolism. Although the molecular mechanisms responsible for its transport across cell membranes remain poorly understood, classical amino acid transport system N appears particularly important. Using intracellular pH measurements, we have now identified an orphan protein related to a vesicular neurotransmitter transporter as system N. Functional analysis shows that this protein (SN1) involves H+ exchange as well as Na+ cotransport and, under physiological conditions, mediates glutamine efflux as well as uptake. Together with the pattern of SN1 expression, these unusual properties suggest novel physiological roles for system N in nitrogen metabolism and synaptic transmission.     

钙对蓝藻固氮受氯化钠胁迫的缓解效应及其与生理条件的关系

氯化钠胁迫导致蓝藻固氮活性的下降,可因加人适当浓度的氯化钠而有一定程度的缓解．在光合作用受抑（暗处理或添加光合抑制剂）、厌氧（Ａｒ或Ｎ２中）和有分子氧的情况下,此种缓解作用减弱．光合作用、需氧代谢（通气）和羟化反应（同时供给氢和氧）正常进行以及碳架（添加外源蔗糖或提高ＣＯ２浓度）供应良好时,钙对氯化钠胁迫的缓解效应增大．改善合成固氮酶的物质基础供应（同时供应ＣＯ２和Ｎ２）对此也有一定的正效应．     

Proteomic Analysis of Low Nitrogen Stress-Responsive Proteins in Roots of Rice

In recent years, the application of proteomic approaches as a tool for global expression analysis and protein identification has been highly efficient in the field of plant research. A solution culture experiment involving two nitrogen treatments, 0.14 mM NH₄NO₃ (low nitrogen (N)) and 1.07 mM NH₄NO₃ (control), was conducted to investigate the response of rice root to low N stress. Root system architecture changed markedly under low N stress, with more lateral roots occurring on the lower part of adventitious roots and longer lateral roots on the upper part, compared to the control. A proteomic approach was employed to further study the rice responses to low N stress. Proteins extracted from roots were profiled by two-dimensional gel electrophoresis, and differentially expressed proteins were analyzed by mass spectrometry. Twelve protein spots were successfully identified by mass spectrometry, 11 of which had known functions. Of these, four were involved with the tricarboxylic acid cycle, two with adenylate metabolism, two with phenylpropanoid metabolism, and two with protein degradation. These differentially expressed proteins play an important role in the responsive mechanisms of rice root to low N stress, and uncovering how the rice proteins respond to low N stress could contribute to improving the nitrogen use efficiency.     

Nitrogen-fixing activity in peat soils from a raised bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014-0.022 mg N/(kg h). Incorporation of the 15N2 isotope into organic compounds of peat soil was from 2.71-8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10-20 cm layer of soil and much lower in the upper (under green moss) and deeper (20-30 cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18-26 h. The maximum nitrogenase activity (3.5-3.8 mg N/(kg h)) observed after 60-70 h coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity without a lag period to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35 degrees C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0-2.5 x 10(6) cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.