Role In Nitrogen Assimilation Research Articles

Effect of thermophilic microbial agents on nitrogen transformation, nitrogen functional genes, and bacterial communities during bean dregs composting.

This study explored how a thermophilic microbial agent altered nitrogen transformation, nitrogen functional genes, and bacterial communities during bean dregs composting with (T) and without (CK) a thermophilic microbial agent for 15days. The results showed that the maximum temperature in T reached 73°C and remained above 70°C for 8days, while that in CK was only 65°C. The pH in T had essentially stabilized on day 7, while that in CK was still increasing. On day 15, the seed germination index (GI) of T (95%) reached maturity (defined by GI ≥ 85%), while the GI of CK was only 36%. The concentrations of total nitrogen, water-soluble nitrogen, ammonia nitrogen, and nitrate nitrogen in T (2.5%, 18.9g/kg, 8.75g/kg, and 1.69g/kg) were all lower than those in CK (3.6%, 28.9g/kg, 12.75g/kg, and 6.82g/kg). During composting, Bacillus played a major role in nitrogen reduction, nitrogen mineralization, denitrification, and the conversion between nitrite and nitrate. Weissella played a major role in nitrogen assimilation. Komagataeibacter and Bacillus played a major role in nitrogen fixation in CK and T, respectively. Nitrification was not observed during composting. The nosZ gene, which converts nitrous oxide to nitrogen, was found only in T. Network analysis suggested that the average number of neighbours in T was 3.30% higher than that in CK and the characteristic path length in T was 14.15% higher than that in CK. Therefore, the thermophilic microbial agents could cause nitrogen loss but promote the maturity of bean dregs, which have great potential application.

Transcriptome Analysis Unveils Gln3 Role in Amino Acids Assimilation and Fluconazole Resistance in Candida glabrata.

After Candida albicans, Candida glabrata is one of the most common fungal species associated with candidemia in nosocomial infections. Rapid acquisition of nutrients from the host is important for the survival of pathogens which possess the metabolic flexibility to assimilate different carbon and nitrogen compounds. In Saccharomyces cerevisiae, nitrogen assimilation is controlled through a mechanism known as Nitrogen Catabolite Repression (NCR). NCR is coordinated by the action of four GATA factors; two positive regulators, Gat1 and Gln3, and two negative regulators, Gzf3 and Dal80. A mechanism in C. glabrata similar to NCR in S. cerevisiae has not been broadly studied. We previously showed that in C. glabrata, Gln3, and not Gat1, has a major role in nitrogen assimilation as opposed to what has been observed in S. cerevisiae in which both factors regulate NCR-sensitive genes. Here, we expand the knowledge about the role of Gln3 from C. glabrata through the transcriptional analysis of BG14 and gln3Δ strains. Approximately, 53.5% of the detected genes were differentially expressed (DEG). From these DEG, amino acid metabolism and ABC transporters were two of the most enriched KEGG categories in our analysis (Up-DEG and Down-DEG, respectively). Furthermore, a positive role of Gln3 in AAA assimilation was described, as was its role in the transcriptional regulation of ARO8. Finally, an unexpected negative role of Gln3 in the gene regulation of ABC transporters CDR1 and CDR2 and its associated transcriptional regulator PDR1 was found. This observation was confirmed by a decreased susceptibility of the gln3Δ strain to fluconazole.

Nitrogen Regulating the Expression and Localization of Four Glutamine Synthetase Isoforms in Wheat (Triticum aestivum L.).

Glutamine synthetase (GS), the key enzyme in plant nitrogen assimilation, is strictly regulated at multiple levels, but the most relevant reports focus on the mRNA level. Using specific antibodies as probes, the effects of nitrogen on the expression and localization of individual wheat GS (TaGS) isoforms were studied. In addition to TaGS2, TaGS1;1 with high affinity to substrate and TaGS1;3 with high catalytic activity were also localized in mesophyll, and may participate in cytoplasmic assimilation of ammonium (NH4+) released from photorespiration or absorbed by roots; TaGS1;2 was localized in xylem of leaves. In roots, although there were hundreds of times more TaGS1;1 than TaGS1;2 transcripts, the amount of TaGS1;1 subunit was not higher than that of TaGS1;2; NH4+ inhibited TaGS1;1 expression but stimulated TaGS1;3 expression. In root tips, nitrate stimulated TaGS1;1, TaGS1;3, and TaGS2 expression in meristem, while NH4+ promoted tissue differentiation and TaGS1;2 expression in endodermis and vascular tissue. Only TaGS1;2 was located in vascular tissue of leaves and roots, and was activated by glutamine, suggesting a role in nitrogen transport. TaGS1;3 was induced by NH4+ in root endodermis and mesophyll, suggesting a function in relieving NH4+ toxicity. Thus, TaGS isoforms play distinct roles in nitrogen assimilation for their different kinetic properties, tissue locations, and response to nitrogen regimes.

Towards identification of molecular mechanism in which the overexpression of wheat cytosolic and plastid glutamine synthetases in tobacco enhanced drought tolerance

Glutamine synthetases (GS) play an essential role in Nitrogen assimilation. Nonetheless, information respecting the molecular functions of GS in drought tolerance (DT) is limited. Here we show that overexpressing cytosolic GS1 or plastidic GS2 gene in tobacco enhanced DT of both root and leaf tissues of the two transgenic seedlings (named as GS1-TR and GS2-TR). RNA-seq analysis on root tissues showed that 83 aquaporin (AQP) genes were identified. Among them, 37 differential expression genes (DEGs) were found in the GS1-TR roots under normal condition, and all were down-regulated; no any DEGs in the GS2-TR roots were found. Contrastingly, under drought, 28 and 32 DEGs of AQP were up-regulated in GS1-TR and GS2-TR roots, respectively. GC-MS analysis on leaf tissues showed that glutamine (Gln) concentrations were negatively correlated AQP expressions in the all four conditions, which suggests that Gln, as a signal molecule, can negatively regulate many AQP expressions. Prestress accumulation of sucrose and proline (Pro) and chlorophyll, and had higher activities of ROS scavengers also contribute the plant DT in both of the two transgenic plants under drought. In addition, 5-aminolevulinic acid (ALA) was up-accumulated in GS2-TR leaves solely under normal condition, which leads to its net photosynthetic rate higher than that in GS1-TR leaves. Last but not the less, the PYL-PP2C-SnRK2 core ABA-signaling pathway was uniquely activated in GS1-TR independent of drought stress (DS). Therefore, our results suggest a possible model reflecting how overexpression of wheat TaGS1 and TaGS2 regulate plant responses to drought.

Growth and secretome analysis of possible synergistic interaction between green algae and cyanobacteria

Synergistic coexistence of nitrogen fixing cyanobacteria such as Anabaena variabilis, Nostoc muscorum and Westiellopsis prolifica with green algae namely Scenedesmus obliquus, Chlorella vulgaris and Botryococcus braunii was studied under nitrogen deficient conditions. The effect of these interactions was investigated on growth, fixed nitrogen content, lipid content and their secretomes in individual cultures and cocultures. Based on the cocultivation studies, it was found that out of the nine interactions studied, B. braunii-N. muscorum synergism was best established. This interaction resulted in a maximum of 50% enhancement in nitrogen fixation in B. braunii-N. muscorum co-culture leading to 27% enhancement in lipid content (membrane and neutral lipid). In general, B. braunii co-cultures showed an enhancement in biomass content of up to 38%. Secretome analysis showed presence of new and modified secondary metabolites having roles in quorum sensing/quenching, interspecies signaling, N-fixation, carbon metabolism, lipid metabolism, antimicrobial activity. Compounds such as trichloroacetic acid and hexadecane were identified that are known to have roles in nitrogen assimilation and carbon metabolism, respectively, were present in some of the co-culture secretomes. The combination of B. braunii-N. muscorum led to the formation of new compounds such as triacontanol which have role in improvement of glucose-lipid metabolism and 9-octadecenamide that is known to be a phytohormone.

Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen–Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies

Plants assimilate inorganic nitrogen absorbed from soil into organic forms as Gln and Glu through the glutamine synthetase/glutamine:2-oxoglutarate amidotransferase (GS/GOGAT) cycle. Whereas GS catalyzes the formation of Gln from Glu and ammonia, GOGAT catalyzes the transfer of an amide group from Gln to 2-oxoglutarate to produce two molecules of Glu. However, the regulatory role of the GS/GOGAT cycle in the carbon–nitrogen balance is not well understood. Here, we report the functional characterization of rice ABNORMAL CYTOKININ RESPONSE 1 (ABC1) gene that encodes a ferredoxin-dependent (Fd)-GOGAT. The weak mutant allele abc1-1 mutant shows a typical nitrogen-deficient syndrome, whereas the T-DNA insertional mutant abc1-2 is seedling lethal. Metabolomics analysis revealed the accumulation of an excessive amount of amino acids with high N/C ratio (Gln and Asn) and several intermediates in the tricarboxylic acid cycle in abc1-1, suggesting that ABC1 plays a critical role in nitrogen assimilation and carbon–nitrogen balance. Five non-synonymous single-nucleotide polymorphisms were identified in the ABC1 coding region and characterized as three distinct haplotypes, which have been highly and specifically differentiated between japonica and indica subspecies. Collectively, these results suggest that ABC1/OsFd-GOGAT is essential for plant growth and development by modulating nitrogen assimilation and the carbon–nitrogen balance.

Gln3 is a main regulator of nitrogen assimilation in Candida glabrata.

After Candida albicans, the yeast Candida glabrata ranks second as an aetiological agent of candidaemia and is the most frequently encountered non-Candida albicans species in patients with invasive candidiasis. Transcriptome analysis in C. albicans, C. glabrata and Cryptoccocus neoformans has revealed that, when engulfed by macrophages, these yeasts upregulate genes involved in nutrient acquisition, including nitrogen transporters such as the general amino acid permease Gap1, the dicarboxylic amino acid permease Dip5, the basic amino acid permease Can1 and the ammonium permeases Mep1 and Mep2. Nitrogen assimilation has been well studied in model species of fungi, such as Aspergillus nidulans, Neurospora crassa and Saccharomyces cerevisiae. However, little is known about nitrogen assimilation in C. glabrata. In the present study, we report a major role for Gln3 in the assimilation of glutamine, ammonium and proline. Ure2 also has a role in nitrogen assimilation, but it is only observable in ammonium and glutamine. In addition, Gat1 has a minor role, which is only observable in the absence of Ure2 and Gln3. Gln3 is absolutely necessary for full ammonium uptake from media. We have also shown that MEP2 gene expression in C. glabrata is completely dependent on Gln3, whereas GAP1 regulation is mainly exerted by Gln3, with the exception of proline where Gat1 has a minor role. In addition, in C. glabrata Ure2 appears to be a negative regulator of these NCR-sensitive genes, similarly to what has been described in S. cerevisiae. Our data place Gln3 as a key regulator of nitrogen assimilation.

Overexpressing a FungalCeGDHGene Improves Nitrogen Utilization and Growth in Rice

ABSTRACTIn lower organisms, NADP(H)‐dependent glutamate dehydrogenases (GDHs) have high affinities to ammonium and play important roles in nitrogen assimilation. Glutamate dehydrogenases in higher plants have lower affinities for ammonium so their nitrogen is mainly assimilated as ammonium by the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway. In present study, an NADP(H)‐dependent glutamate dehydrogenase gene (CeGDH) was cloned from the fungusCylindrocarpon ehrenbergii(Ce) and ectopically expressed in rice (Oryza sativaL. ‘Kitaake’). Meanwhile, the enzyme activity ofCeGDH was detected and the effects ofCeGDH in transgenic rice were analyzed. The determination of enzymatic activity in vitro showed thatCeGDH's aminating activity is higher than its deaminating activity, which was also confirmed by the results of enzymatic activity in vivo inCeGDHtransgenic rice. These results suggested that theCeGDH is inclined to convert 2‐Oxoglutarate to glutamate. Additionally, the nitrogen assimilation ability and grain yield were analyzed inCeGDHtransgenic plants. The hydroponic experiments revealed that the shoot and root lengths and nitrogen contents were improved obviously in transgenic plants compared with the wild‐type under low‐nitrogen conditions at seedling stage. In field conditions, the agronomic traits analysis showed that panicle numbers and grain yields of the transgenic plants also increased significantly under low‐nitrogen conditions. These results demonstrated that the introduction ofCeGDHinto rice could enhance nitrogen utilization, improve growth, and increase grain yields, especially in low nitrogen fertility.

The roles of glutamate dehydrogenase, glutamine synthetase and three forms of glutamate synthase on nitrogen assimilation in various organs of Pisum arvense L.

The activities of GDH, GS and three forms of GOGAT (NADH, NADPH or ferredoxin-dependent) were studied in the leaves, stems and roots of the <i>Pisum arvense</i>. GS and the individual forms of GOGAT dominated in the leaves of 7 day-old plants which were taking up NO<sub>3</sub><sup>-</sup> or NH<sub>4</sub><sup>+</sup> ions, while NADH-GDH dominated in the roots of these plants. In comparison with HNO<sub>3</sub><sup>-</sup> , NH<sub>4</sub><sup>+</sup> ions stimulated the activity of most of the enzymes of the GS/GOGAT and GDH pathways in stems and roots, while in and leaves this effect was age-dependent. The Fd-GOGAT located in leaves and stems was not regulated by NH<sub>4</sub><sup>+</sup> , which indicates that this enzyme is not likely to be directly involved in the assimilation of NH<sub>4</sub><sup>+</sup> ions that have been taken up. The obtained data indicate that at lower tissue NH<sub>4</sub><sup>+</sup> concentration a decisive role in nitrogen assimilation in leaves and stems is played by the GS/GOGAT pathway, while in the roots-by GDH and in less degree by GS, GOGAT. High amounts of accumulated NH<sub>4</sub><sup>+</sup> ions set off a detoxication mechanism which includes NADH-GDH, common to all tissues. Only in 7 day-old leaves did the detoxication of NH<sub>4</sub><sup>+</sup> take place with the involvement of NADH-GOGAT and NADPH-GOGAT.

The sinks of dissolved inorganic nitrogen in surface water of wetland mesocosms

The transport of dissolved inorganic nitrogen in wetlands was investigated in the Yeyahu National Preserve of Beijing, China, from July to December 2011. Stable isotope of nitrogen (15N) as NH4+ was added to experimental mesocosms vegetated by seven common wetland plants. The 15N abundance in soil and various plant parts were measured at prescribed time intervals during the 49-day experimental period. Rapid 15N enrichments were found in soil and aquatic plants during the first sampling event at day 3 and typically increased linearly during the experimental period. Highest 15N enrichment was found in submerged plants, followed by emergent plants and soil. No differences in δ15N values were found among leaves and stems of all submerged plants (Potamogeton pusillus, Potamogeton perfoliatus, Najas marina, Ceratophyllum demersum), suggesting that these plant structures are equally effective in nitrogen assimilation. The roots of P. perfoliatus displayed the lowest enrichment compared to the stems and leaves. Such results indicate that submerged plant roots may have a relatively weak role in nitrogen assimilation. The two emergent plants (Phragmites australis and Typha orientalis) showed no differences in δ15N values among all plant parts, suggesting rapid translocation of nitrogen from roots to other portions. This experiment demonstrates that wetland soil and plant stems and leaves are major sinks for dissolved nitrogen from the surface water and provides valuable insight into nitrogen retention and transport pathways in various wetland plants.

Overexpressing the ZmDof1 gene in Populus does not improve growth and nitrogen assimilation under low-nitrogen conditions

Genetic engineering of nitrogen assimilation has received considerable attention for its improvement of nitrogen use efficiency in crops. Overexpression of maize Dof1 (ZmDof1), a plant-specific transcription factor, has been reported to promote nitrogen assimilation and growth under low-nitrogen conditions in transgenic Arabidopsis and rice. The present study explored its application in Populus by overexpressing the ZmDof1 gene. Contrary to the results in Arabidopsis and rice, introduction of the ZmDof1 gene did not improve growth of transgenic Populus under different low-nitrogen conditions both in vitro and in greenhouse. Expressions of genes that are involved in nitrogen assimilation and carbon fixation were not induced in ZmDof1-transgenic Populus. Nitrogen/carbon concentrations and photosynthesis rate were not increased in the ZmDof1-transgenic Populus. The results in transgenic Populus overexpressing the ZmDof1 gene indicated that ZmDof1 did not play an important role in nitrogen assimilation under low nitrogen condition in woody Populus plants as it did in annual Arabidopsis and rice plants.

Mutagenesis and Functional Characterization of the Four Domains of GlnD, a Bifunctional Nitrogen Sensor Protein

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) protein; P(II) in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

Fatty Acid-amino Acid Conjugates Diversification in Lepidopteran Caterpillars

Fatty acid amino acid conjugates (FACs) have been found in noctuid as well as sphingid caterpillar oral secretions; in particular, volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and its biochemical precursor, N-linolenoyl-L-glutamine, are known elicitors of induced volatile emissions in corn plants. These induced volatiles, in turn, attract natural enemies of the caterpillars. In a previous study, we showed that N-linolenoyl-L-glutamine in larval Spodoptera litura plays an important role in nitrogen assimilation which might be an explanation for caterpillars synthesizing FACs despite an increased risk of attracting natural enemies. However, the presence of FACs in lepidopteran species outside these families of agricultural interest is not well known. We conducted FAC screening of 29 lepidopteran species, and found them in 19 of these species. Thus, FACs are commonly synthesized through a broad range of lepidopteran caterpillars. Since all FAC-containing species had N-linolenoyl-L-glutamine and/or N-linoleoyl-L-glutamine in common, and the evolutionarily earliest species among them had only these two FACs, these glutamine conjugates might be the evolutionarily older FACs. Furthermore, some species had glutamic acid conjugates, and some had hydroxylated FACs. Comparing the diversity of FACs with lepidopteran phylogeny indicates that glutamic acid conjugates can be synthesized by relatively primitive species, while hydroxylation of fatty acids is limited mostly to larger and more developed macrolepidopteran species.

Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis

BackgroundThe assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria. The physiological role and pathogenic properties of glutamine synthetase (GS) have been extensively investigated in Mycobacterium tuberculosis. However, little is known about this enzyme in other mycobacterial species, or the role of an additional nitrogen assimilatory pathway via glutamate dehydrogenase (GDH), in the mycobacteria as a whole. We investigated specific enzyme activity and transcription of GS and as well as both possible isoforms of GDH (NAD+- and NADP+-specific GDH) under varying conditions of nitrogen availability in Mycobacterium smegmatis as a model for the mycobacteria.ResultsIt was found that the specific activity of the aminating NADP+-GDH reaction and the deaminating NAD+-GDH reaction did not change appreciably in response to nitrogen availability. However, GS activity as well as the deaminating NADP+-GDH and aminating NAD+-GDH reactions were indeed significantly altered in response to exogenous nitrogen concentrations. Transcription of genes encoding for GS and the GDH isoforms were also found to be regulated under our experimental conditions.ConclusionsThe physiological role and regulation of GS in M. smegmatis was similar to that which has been described for other mycobacteria, however, in our study the regulation of both NADP+- and NAD+-GDH specific activity in M. smegmatis appeared to be different to that of other Actinomycetales. It was found that NAD+-GDH played an important role in nitrogen assimilation rather than glutamate catabolism as was previously thought, and is it's activity appeared to be regulated in response to nitrogen availability. Transcription of the genes encoding for NAD+-GDH enzymes seem to be regulated in M. smegmatis under the conditions tested and may contribute to the changes in enzyme activity observed, however, our results indicate that an additional regulatory mechanism may be involved. NADP+-GDH seemed to be involved in nitrogen assimilation due to a constitutive aminating activity. The deaminating reaction, however was observed to change in response to varying ammonium concentrations which suggests that NADP+-GDH is also regulated in response to nitrogen availability. The regulation of NADP+-GDH activity was not reflected at the level of gene transcription thereby implicating post-transcriptional modification as a regulatory mechanism in response to nitrogen availability.

Active role of fatty acid amino acid conjugates in nitrogen metabolism in Spodoptera litura larvae

Since the first fatty acid amino acid conjugate (FAC) was isolated from regurgitant of Spodoptera exigua larvae in 1997 [volicitin: N-(17-hydroxylinolenoyl)-L-glutamine], their role as elicitors of induced responses in plants has been well documented. However, studies of the biosyntheses and the physiological role of FACs in the insect have been minimal. By using (14)C-labeled glutamine, glutamic acid, and linolenic acid in feeding studies of Spodoptera litura larvae, combined with tissue analyses, we found glutamine in the midgut cells to be a major source for biosynthesis of FACs. Furthermore, 20% of the glutamine moiety of FACs was derived from glutamic acid and ammonia through enzymatic reaction of glutamine synthetase (GS). To determine whether FACs improve GS productivity, we studied nitrogen assimilation efficiency of S. litura larvae fed on artificial diets containing (15)NH(4)Cl and glutamic acid. When the diet was enriched with linolenic acid, the nitrogen assimilation efficiency improved from 40% to >60%. In the lumen, the biosynthesized FACs are hydrolyzed to fatty acids and glutamine, which are reabsorbed into tissues and hemolymph. These results strongly suggested that FACs play an active role in nitrogen assimilation in Lepidoptera larva and that glutamine containing FACs in the gut lumen may function as a form of storage of glutamine, a key compound of nitrogen metabolism.

Functional Analysis of GlnE, an Essential Adenylyl Transferase in Mycobacterium tuberculosis

Glutamine synthetase (GS) plays an important role in nitrogen assimilation. The major GS of Mycobacterium tuberculosis is GlnA1, a type I GS whose activity is controlled by posttranscriptional modification by GlnE. GlnE is an adenylyl transferase comprised of an adenylylating domain and a deadenylylating domain which modulate GS activity. We previously demonstrated that GlnE is essential in M. tuberculosis in normal growth medium. In this study, we further show that GlnE is required under multiple medium conditions, including in nitrogen-limited medium. We demonstrate that adenylylation is the critical activity for M. tuberculosis survival, since we were able to delete the deadenylylation domain with no apparent effect on growth or GS activity. Furthermore, we identified a critical aspartate residue in the proposed nucleotidyltransferase motif. Temperature-sensitive mutants of GlnE were generated and shown to have a defect in growth and GS activity in nitrogen-limited medium. Finally, we were able to generate a GlnE null mutant in the presence of L-methionine sulfoximine, a GS inhibitor, and glutamine supplementation. In the presence of these supplements, the null mutant was able to grow similarly to the wild type. Surprisingly, the GlnE mutant was able to survive and grow for extended periods in liquid medium, but not on solid medium, in the absence of GS inhibition. Thus, we have confirmed that the unusual requirement of M. tuberculosis for GlnE adenylylation activity is linked to the activity of GS in the cell.

Growth Phase-dependent Activation of Nitrogen-related Genes by a Control Network of Group 1 and Group 2 σ Factors in a Cyanobacterium

It has been reported that an RNA polymerase sigma factor, SigC, mainly contributes to specific transcription from the promoter PglnB-54,-53 under nitrogen-deprived conditions during the stationary phase of cell growth in the cyanobacterium Synechocystis sp. strain PCC 6803 (Asayama, M., Imamura, S., Yoshihara, S., Miyazaki, A., Yoshida, N., Sazuka, T., Kaneko, T., Ohara, O., Tabata, S., Osanai, T., Tanaka, K., Takahashi, H., and Shirai, M. (2004) Biosci. Biotechnol. Biochem. 68, 477-487). In this study, we further examined the functions of group 2 sigma factors of RNA polymerase in NtcA-dependent nitrogen-related gene expression in PCC 6803. Results indicated that SigB and SigC contribute to the transcription from PglnB-54,-53 with a sigma factor replaced in a growth phase-dependent manner. We also confirmed the contribution of SigB and SigC to the transcription of other NtcA-dependent genes, glnA, sigE, and amt1, as in the case of glnB. On the other hand, the transcription of glnN was dependent on SigB and SigE. In the SigB and SigC-based regulation, the level of SigB increased, but that of SigC was constant under conditions of nitrogen deprivation. Furthermore, it was found that SigC negatively and positively regulates the level of SigB in the log and stationary phase, respectively. SigC also had a positive effect on the level of sigB transcript during the stationary phase. In contrast, SigB acts positively on SigC levels in both growth phases. These results and previous findings indicated that multiple group 2 sigma factors take part in the control of NtcA-dependent nitrogen-related gene expression in cooperation with a group 1 sigma factor, SigA.

GlnD is essential for NifA activation, NtrB/NtrC-regulated gene expression, and posttranslational regulation of nitrogenase activity in the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme and is thought to be the primary sensor of nitrogen status in the cell. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) proteins, which in turn regulate a variety of other proteins. We report here the characterization of glnD mutants from the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum and the analysis of the roles of GlnD in the regulation of nitrogen fixation. Unlike glnD mutations in Azotobacter vinelandii and some other bacteria, glnD deletion mutations are not lethal in R. rubrum. Such mutants grew well in minimal medium with glutamate as the sole nitrogen source, although they grew slowly with ammonium as the sole nitrogen source (MN medium) and were unable to fix N(2). The slow growth in MN medium is apparently due to low glutamine synthetase activity, because a DeltaglnD strain with an altered glutamine synthetase that cannot be adenylylated can grow well in MN medium. Various mutation and complementation studies were used to show that the critical uridylyltransferase activity of GlnD is localized to the N-terminal region. Mutants with intermediate levels of uridylyltransferase activity are differentially defective in nif gene expression, the posttranslational regulation of nitrogenase, and NtrB/NtrC function, indicating the complexity of the physiological role of GlnD. These results have implications for the interpretation of results obtained with GlnD in many other organisms.

Glutamine synthetase of potato (Solanum tuberosum L. cv. Desiree) plants: cell- and organ-specific expression and differential developmental regulation reveal specific roles in nitrogen assimilation and mobilization

Potato (Solanum tuberosum L. cv. Desiree) glutamine synthetase (GS) (EC 6.3.1.2) gene expression and polypeptide accumulation patterns were analysed in several organs and at several developmental stages. Three GS genes have been identified, one gene encoding plastidic GS (GS2) and two encoding cytosolic GS (GS1) that are differentially expressed in the plant at cellular and organ levels. Specific developmental regulation of different GS genes was also observed. Potato GS seems to be regulated essentially at transcription and/or RNA stability levels. GS2 polypeptides and mRNAs were detected in leaves and their content decreased as leaves senesced. A similar pattern of expression was observed for the GS1 gene Stgs1a. GS1 transcripts and polypeptides were present in all organs analysed and are the only GS detected in non-photosynthetic tissues and in the later leaf senescing stages. The increase in GS1 during leaf senescence mainly reflected polypeptide and transcript accumulation of the GS1-encoding gene Stgs1b. In situ hybridization results point to a cell-specific expression of GS1 genes within the vascular bundles, Stgs1b being expressed in the xylem and phloem parenchyma cells, and Stgs1a being expressed only in the phloem companion cells. This pattern of spatial distribution and differential developmental regulation of different GS1 genes differs from what has been previously described for genes of other Solanaceae with a high degree of similarity with the ones described here and suggests that distinct GS1 isozymes have specific and possibly distinct roles within the same organ. These new findings highlight the physiological importance of different GS1 isoenzymes in plant nitrogen metabolism.

Dynamics of the Nitrogen-Efficient Guild and Its Relationship to Nitrogen and Carbon Patterns in Two Desert Soil Ecosystems

Nitrogen fixation is generally considered to play a major role in nitrogen assimilation in desert soils, and the Nitrogen-Efficient Guild (NEG) is a useful index of the free-living nitrogen fixer population. Nitrogen-Efficient Guild dynamics in macrophytic patches in the Negev Desert probably contribute to the understanding of nitrogen processes in desert ecosystems. It is also important to determine the relationship between the Nitrogen-Efficient Guild numbers and nutrient availability on a tempospatial dynamic basis. This study was conducted in the Negev Desert during a period of one year. During this time, the Nitrogen-Efficient Guild number fluctuated between 3.9 - 0.7 2 10 6 and 28.5 - 5.3 2 10 6 colony forming units (CFU) g -1 in a loessial soil site, and 0.7 - 0.2 2 10 6 and 10.7 - 1.8 2 10 6 in a sandy soil site, with significant peak values (P < 0.05) during the winter season. The results demonstrate that significantly larger populations of free-living nitrogen fixers exist in soils under shrub canopies, suggesting considerable nitrogen fixation ability. It was also concluded that the nitrogen-fixing populations and communities vary with seasons in the desert, since they are mainly controlled by soil moisture. Furthermore, the Nitrogen-Efficient Guild numbers exhibit a significant correlation with total organic carbon and total soluble nitrogen, but not with dissolved organic carbon.