Nitrogen Metabolite Research Articles

RrmAregulates the stability of specific transcripts in response to both nitrogen source and oxidative stress

Differential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well-characterized example is glutamine signalled degradation of specific transcripts in Aspergillus nidulans. In the case of areA, which encodes a wide-domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3′ UTR. Utilizing this RNA sequence we isolated RrmA, an RNA recognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization of niaD mRNA. However, nitrogen starvation was shown to act independently of RrmA in stabilizing certain transcripts. RrmA was also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmA mutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization of eifE and dhsA mRNA. eifE encodes eIF5A, a translation factor within which a conserved lysine is post-translationally modified to hypusine, a process requiring DhsA. Intriguingly, for specific transcripts RrmA mediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts.

Pseudo-constitutivity of nitrate-responsive genes in nitrate reductase mutants

In fungi, transcriptional activation of genes involved in NO3- assimilation requires the presence of an inducer (nitrate or nitrite) and low intracellular concentrations of the pathway products ammonium or glutamine. In Aspergillus nidulans, the two transcription factors NirA and AreA act synergistically to mediate nitrate/nitrite induction and nitrogen metabolite derepression, respectively. In all studied fungi and in plants, mutants lacking nitrate reductase (NR) activity express nitrate-metabolizing enzymes constitutively without the addition of inducer molecules. Based on their work in A. nidulans, Cove and Pateman proposed an “autoregulation control” model for the synthesis of nitrate metabolizing enzymes in which the functional nitrate reductase molecule would act as co-repressor in the absence and as co-inducer in the presence of nitrate. However, NR mutants could simply show “pseudo-constitutivity” due to induction by nitrate which accumulates over time in NR-deficient strains. Here we examined this possibility using strains which lack flavohemoglobins (fhbs), and are thus unable to generate nitrate internally, in combination with nitrate transporter mutations (nrtA, nrtB) and a GFP-labeled NirA protein. Using different combinations of genotypes we demonstrate that nitrate transporters are functional also in NR null mutants and show that the constitutive phenotype of NR mutants is not due to nitrate accumulation from intracellular sources but depends on the activity of nitrate transporters. However, these transporters are not required for nitrate signaling because addition of external nitrate (10mM) leads to standard induction of nitrate assimilatory genes in the nitrate transporter double mutants. We finally show that NR does not regulate NirA localization and activity, and thus the autoregulation model, in which NR would act as a co-repressor of NirA in the absence of nitrate, is unlikely to be correct. Results from this study instead suggest that transporter-mediated NO3- accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed “pseudo-constitutive”.

Nitrogen metabolites in cucumber leaves treated with parsley root extracts

以黄瓜品种"津春4号"为材料, 在黄瓜植株第1片真叶长至宽5 cm时, 使用西芹鲜根及根际区物的乙醇、丙酮和蒸馏水浸提液对黄瓜植株进行化感处理(灌根), 测定处理后黄瓜叶片内几种氮代谢物质含量的变化, 从氮代谢角度研究西芹鲜根及根际区物浸提液处理后黄瓜植株对黄瓜枯萎病菌的化感抑制作用机理。结果表明: 处理后黄瓜叶片内可溶性蛋白质、氨、丝氨酸、精氨酸、脯氨酸与总氨基酸含量均高于对照, 含量达到峰值时分别较丙酮对照增加10.58%、18.31%、6.54%、3.10%、9.64%和3.16%, 而硝酸盐、甘氨酸和蛋氨酸含量则低于对照, 含量达到最低值时分别较丙酮对照降低7.52%、3.41%和15.55%。对于不同西芹根物质浸提液处理, 丙酮浸提液处理的氮代谢物质含量变化最显著, 乙醇浸提液处理次之, 蒸馏水浸提液处理变化最低, 西芹鲜根浸提液处理的氮代谢物质含量变化高于西芹根际区物浸提液处理。

Characterization of an Nmr Homolog That Modulates GATA Factor-Mediated Nitrogen Metabolite Repression in Cryptococcus neoformans

Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis.

Nitrogen metabolite repression of metabolism and virulence in the human fungal pathogen Cryptococcus neoformans.

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine-three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°-40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection.

Crystal Structures of NAD(P)(H) Sensing NmrA-Like Proteins from Dictyostelium Discoideum

NAD(P)(H) functions as central coenzyme in catabolic and anabolic reaction. And recently, many results show that NAD(P)(H) is also involved in signaling pathways that regulate fundamental processes of cellular functions. NmrA in Aspergillus nidulans is known to discriminate between oxidized and reduced dinucleotides and suggested as redox sensor. There are several NmrA-like protein coding genes in Dictyostelium discoideum and they show developmental stage-dependent expression levels and especially DDB_G0291732 gene exhibits different expression pattern from the other NmrA-like protein coding genes. To study relation between structure and function, the crystal structures of DDB_G0291732 and DDB_G0286605 were determined. Crystal structures of DDB_G0291732 and DDB_G0286605 adopt the features of SDR-type NmrA-like proteins. DDB_G0291732 and DDB_G0286605 contain conserved N-terminal dinucleotide binding Rossmann fold with a central β sheet and the C-domain, containing a hydrophobic pocket connected to the N-domain. Structural comparisons with known 3D structures reveal that DDB_G0291732 and DDB_G0286605 are mostly matched with HSCARG; human NADP(H) sensor protein, NmrA; negative transcriptional regulator involved in nitrogen metabolite repression of Aspergillus nidulans and QOR2; new-type quinone oxidoreductase considered to function as transcriptional regulator of E. coli. No dehydrogenase activity could be prospected with DDB_G0291732 and DDB_G0286605 because the catalytic Tyr residue in the active site triad of Ser-Tyr-Lys is replaced by His and Asp in DDB_G0291732 and DDB_G0286605, respectively. Cofactor binding experiments reveal that DDB_G0291732 does not interact with pyridine dinucleotides and DDB_G0286605 has high affinity with NADP(H) but not with NAD(H). The mRNA expression levels of DDB_G0291732 and DDB_G0286605 genes after dehydroepiandrosterone (DHEA) or H2O2 treatment are monitored to examine how these proteins respond to redox states in D. discoideum.

The bZIP Transcription Factor MeaB Mediates Nitrogen Metabolite Repression at Specific Loci

In Fusarium fujikuroi, bikaverin (BIK) biosynthesis is subject to repression by nitrogen. Unlike most genes subject to nitrogen metabolite repression, it has been shown that transcription of bik biosynthetic genes is not AreA dependent. Searching for additional transcription factors that may be involved in nitrogen regulation, we cloned and characterized the orthologue of Aspergillus nidulans meaB, which encodes a bZIP transcription factor. Two transcripts are derived from F. fujikuroi meaB: the large transcript (meaB(L)) predominates under nitrogen-sufficient conditions and the smaller transcript (meaB(S)) under nitrogen limitation, in an AreA-dependent manner. MeaB is specifically translocated to the nucleus under nitrogen-sufficient conditions in both F. fujikuroi and A. nidulans. Deletion of meaB resulted in partial upregulation of several nitrogen-regulated genes, but only in the ΔmeaB ΔareA double mutant were the bikaverin genes significantly upregulated in the presence of glutamine. These data demonstrate that MeaB and AreA coordinately mediate nitrogen metabolite repression and, importantly, that independently of AreA, MeaB can mediate nitrogen metabolite repression at specific loci in F. fujikuroi.

UreA, the major urea/H + symporter in Aspergillus nidulans

We report here the characterization of UreA, a high-affinity urea/H + symporter of Aspergillus nidulans. The deletion of the encoding gene abolishes urea transport at low substrate concentrations, suggesting that in these conditions UreA is the sole transport system specific for urea in A. nidulans. The ureA gene is not inducible by urea or its precursors, but responds to nitrogen metabolite repression, necessitating for its expression the AreA GATA factor. In contrast to what was observed for other transporters in A. nidulans, repression by ammonium is also operative during the isotropic growth phase. The activity of UreA is down-regulated post-translationally by ammonium-promoted endocytosis. A number of homologues of UreA have been identified in A. nidulans and other Aspergilli, which cluster in four groups, two of which contain the urea transporters characterized so far in fungi and plants. This phylogeny may have arisen by gene duplication events, giving place to putative transport proteins that could have acquired novel, still unidentified functions.

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signaling molecules is poorly characterized. The transcription repressor NmrA of Aspergillus nidulans discriminates between oxidized and reduced dinucleotides, however, dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelium grown under nitrogen sufficient conditions but abundant in mycelium starved of nitrogen. One of the proteases was purified and identified as the protein Q5BAR4 encoded by the gene AN2366.2. Fluorescence confocal microscopy showed that the nuclear levels of NmrA were reduced approximately 38% when mycelium was grown on nitrate compared to ammonium and absent when starved of nitrogen. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. NmrA digested at the C-terminal site was unable to bind to the AreA zinc finger. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site retained the ability to bind NAD(+) and showed a resistance to further digestion that was enhanced by the presence of NAD(+). This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated.

CNI1/ATL31, a RING‐type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

Negative Roles of a Novel Nitrogen Metabolite Repression-Related Gene, TAR1 , in Laccase Production and Nitrate Utilization by the Basidiomycete Cryptococcus neoformans

The multicopper oxidase laccase is widespread in fungi and has great industrial importance. One puzzle regarding laccase production in the basidiomycetous yeast Cryptococcus neoformans is that it is inhibited by high temperature (e.g., 37 degrees C). In this paper, we report identification of a nitrogen metabolite repression-related gene, TAR1, which is responsible for laccase repression. Disruption of TAR1 results in a significant increase in the level of LAC1 mRNA at 37 degrees C. The putative protein Tar1 shares a moderate level of similarity with the nitrogen metabolite repressors Nmr1 and NmrA from Neurospora crassa and Aspergillus nidulans, respectively. Likewise, Tar1 has a negative role in the utilization of nitrate. Furthermore, the structure of Tar1 is unique. Tar1 lacks the long C-terminal region of Nmr1 and NmrA. It contains the canonical Rossmann fold motif, GlyXXGlyXXGly, whereas Nmr1 and NmrA have variable residues at the Gly positions. Interestingly, the promoter region of TAR1 contains three TTC/GAA repeats which are likely the heat shock factor (Hsf) binding sites, implying that Hsf has a role in laccase inhibition. TAR1 mediation of temperature-associated repression of LAC1 suggests a novel mechanism of laccase regulation and a new function for Nmr proteins. Our work may be helpful for industry in terms of promotion of laccase activity.

AgtA, the Dicarboxylic Amino Acid Transporter of Aspergillus nidulans , Is Concertedly Down-Regulated by Exquisite Sensitivity to Nitrogen Metabolite Repression and Ammonium-Elicited Endocytosis

We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.

Effects of Fortifying Low Crude Protein Diet with Crystalline Amino Acids on Performance, Blood Ammonia Level, and Excreta Characteristics of Broiler Chicks

A study was conducted in a completely randomized design to evaluate the performance, excreta characteristics, and some blood nitrogen metabolite concentrations of 28-d-old male broilers fed 4 experimental diets in which CP was decreased in a stepwise manner from 23 to 17%. The other 4 diets were formulated to have 19 and 17% CP, in which 2 of them contained an additional 10% of particular essential amino acids (EAA) and 2 were supplemented with Gly and Glu. Ileal digestible quantities of all EAA were almost equal in the diets, and total amount of each EAA was maintained at or above NRC requirements. Decreasing dietary CP below 19% depressed performance and appetite and increased fat deposition in the whole body and abdominal cavity significantly. Adding the Gly and Glu mixtures to low-CP diets improved performance and decreased fat deposition. Uric acid, moisture, and acidity of excreta were decreased by reduction of dietary CP; excretory ammonia level was increased in 17% CP diets. Blood ammonia level was increased and plasma uric acid was decreased with reduction of CP to 17%. Supplementing Gly and Glu increased plasma and excretory uric acid level in spite of decreasing blood ammonia concentration. The aminostatic hypothesis cannot explain the sharp reduction in appetite in this experiment, because alteration of dietary CP had no significant influence on most plasma free amino acid levels. Therefore, reduction of CP to 19% not only does not impair performance but also decrease nitrogen, ammonia, and pH of excreta that may improve upon litter and air quality. Adding large amounts of crystalline EAA to diets with low intact CP increased blood and excretory ammonia concentration, which due to its negative effects on tissue metabolism may be the main cause of retarded growth and appetite in decreased CP diets below 19%.

A systems view of nitrogen nutrient and metabolite responses in Arabidopsis

Nitrogen (N) is an essential macronutrient available to plants mainly as nitrate in agricultural soils. Besides its role as a nutrient, inorganic and organic N sources play key roles as signals that control genome-wide gene expression in Arabidopsis and other plant species. Genomics approaches have provided us with thousands of genes whose expression is modulated in response to N treatments in Arabidopsis. Recently, systems approaches have been utilized to map the complex molecular network that plants utilize to integrate metabolic, cellular, and developmental processes to successfully adapt to changing N availability. The challenge now is to understand the molecular mechanisms underlying N regulation of gene networks and bridge the gap between N sensing, signaling, and downstream physiological and developmental changes. We discuss recent advances in this direction.

A new protein carrying an NmrA-like domain is required for cell differentiation and development in Dictyostelium discoideum

We have isolated a Dictyostelium mutant unable to induce expression of the prestalk-specific marker ecmB in monolayer assays. The disrupted gene, padA, leads to a range of phenotypic defects in growth and development. We show that padA is essential for growth, and we have generated a thermosensitive mutant allele, padA − . At the permissive temperature, mutant cells grow poorly; they remain longer at the slug stage during development and are defective in terminal differentiation. At the restrictive temperature, growth is completely blocked, while development is permanently arrested prior to culmination. padA − slugs are deficient in prestalk A cell differentiation and present an abnormal ecmB expression pattern. Sequence comparisons and predicted three-dimensional structure analyses show that PadA carries an NmrA-like domain. NmrA is a negative transcriptional regulator involved in nitrogen metabolite repression in Aspergillus nidulans. PadA predicted structure shows a NAD(P) +-binding domain, which we demonstrate that is essential for function. We show that padA − development is more sensitive to ammonia than wild-type cells and two ammonium transporters, amtA and amtC, appear derepressed during padA − development. Our data suggest that PadA belongs to a new family of NAD(P) +-binding proteins that link metabolic changes to gene expression and is required for growth and normal development.

Nucleosome Positioning and Histone H3 Acetylation Are Independent Processes in the Aspergillus nidulans prnD-prnB Bidirectional Promoter

In Aspergillus nidulans, proline can be used as a carbon and nitrogen source, and its metabolism requires the integration of three signals, including proline induction and nitrogen and carbon metabolite derepression. We have previously shown that the bidirectional promoter in the prnD-prnB intergenic region undergoes drastic chromatin rearrangements such that proline induction leads to the loss of positioned nucleosomes, whereas simultaneous carbon and nitrogen metabolite repression results in the partial repositioning of these nucleosomes. In the proline cluster, the inhibition of deacetylases by trichostatin A leads to partial derepression and is associated with a lack of nucleosome positioning. Here, we investigate the effect of histone acetylation in the proline cluster using strains deleted of essential components of putative A. nidulans histone acetyltransferase complexes, namely, gcnE and adaB, the orthologues of the Saccharomyces cerevisiae GCN5 and ADA2 genes, respectively. Surprisingly, GcnE and AdaB are not required for transcriptional activation and chromatin remodeling but are required for the repression of prnB and prnD and for the repositioning of nucleosomes in the divergent promoter region. Chromatin immunoprecipitation directed against histone H3 lysines K9 and K14 revealed that GcnE and AdaB participate in increasing the acetylation level of at least one nucleosome in the prnD-prnB intergenic region during activation, but these activities do not determine nucleosome positioning. Our results are consistent with a function of GcnE and AdaB in gene repression of the proline cluster, probably an indirect effect related to the function of CreA, the DNA-binding protein mediating carbon catabolite repression in A. nidulans.

Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence.

Trehalose fulfils a wide variety of functions in cells, acting as a stress protectant, storage carbohydrate and compatible solute. Recent evidence, however, indicates that trehalose metabolism may exert important regulatory roles in the development of multicellular eukaryotes. Here, we show that in the plant pathogenic fungus Magnaporthe grisea trehalose-6-phosphate (T6P) synthase (Tps1) is responsible for regulating the pentose phosphate pathway, intracellular levels of NADPH and fungal virulence. Tps1 integrates glucose-6-phosphate (G6P) metabolism with nitrogen source utilisation, and thereby regulates the activity of nitrate reductase. Activity of Tps1 requires an associated regulator protein Tps3, which is also necessary for pathogenicity. Tps1 controls expression of the nitrogen metabolite repressor gene, NMR1, and is required for expression of virulence-associated genes. Functional analysis of Tps1 indicates that its regulatory functions are associated with binding of G6P, but independent of Tps1 catalytic activity. Taken together, these results demonstrate that Tps1 is a central regulator for integration of carbon and nitrogen metabolism, and plays a pivotal role in the establishment of plant disease.

Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides

Filamentous nitrogen fixing cyanobacteria are key players in global nutrient cycling, but the relationship between CO2- and N2-fixation and intercellular exchange of these elements remains poorly understood in many genera. Using high-resolution nanometer-scale secondary ion mass spectrometry (NanoSIMS) in conjunction with enriched H13CO3- and 15N2 incubations of Anabaena oscillarioides, we imaged the cellular distributions of C, N and P and 13C and 15N enrichments at multiple time points during a diurnal cycle as proxies for C and N assimilation. The temporal and spatial distributions of the newly fixed C and N were highly heterogeneous at both the intra- and inter-cellular scale, and indicative of regions performing active assimilation and biosynthesis. Subcellular components such as the neck region of heterocycts, cell division septae and putative cyanophycin granules were clearly identifiable by their elemental composition. Newly fixed nitrogen was rapidly exported from heterocysts and was evenly allocated among vegetative cells, with the exception of the most remote vegetative cells between heterocysts, which were N limited based on lower 15N enrichment. Preexisting functional heterocysts had the lowest levels of 13C and 15N enrichment, while heterocysts that were inferred to have differentiated during the experiment had higher levels of enrichment. This innovative approach, combining stable isotope labeling and NanoSIMS elemental and isotopic imaging, allows characterization of cellular development (division, heterocyst differentiation), changes in individual cell composition and cellular roles in metabolite exchange.

Metabolite transport across the peribacteroid membrane during broad bean development

A temporal pattern of the peribacteroid membrane (PBM) transport function was studied. Spectrophotometric recording was used for establishing the effect of carbon-and nitrogen-containing substrates (malate, succinate, and glutamate) on the acidification of the peribacteroid space and the intensity of light scattering in the symbiosome suspension from broad bean (Vicia faba L.) root nodules of different age. At the early stages of nodule formation and functioning, PBM is permeable not only for malate and succinate, but also for glutamate, and this permeability fully provides for the active bacteroid division and the nitrogenase complex synthesis in the bacteroids at the expense of the carbon-and nitrogen-containing substrates. Mature nodules are characterized by the greatest nitrogen-fixing activity. In these nodules, PBM is selectively permeable for malate and succinate, but constitutes a barrier for glutamate. Thereby, mutually beneficial relations between the symbiotic partners are achieved. In senescent nodules, a rearrangement of symbiotic interactions is directed toward a minimization of both carbon and nitrogen metabolite consumption by the bacteroids. It is concluded that, in the course of the development of the legume-rhizobia symbiosis, the PBM transport function is changed. This function determines a qualitatively different pattern of symbiotic partner interactions in the following sequence: parasitism-mutualism-commensalism.

Role of the Fusarium fujikuroi TOR Kinase in Nitrogen Regulation and Secondary Metabolism

In Fusarium fujikuroi, the biosynthesis of gibberellins (GAs) and bikaverin is under control of AreA-mediated nitrogen metabolite repression. Thus far, the signaling components acting upstream of AreA and regulating its nuclear translocation are unknown. In Saccharomyces cerevisiae, the target of rapamycin (TOR) proteins, Tor1p and Tor2p, are key players of nutrient-mediated signal transduction to control cell growth. In filamentous fungi, probably only one TOR kinase-encoding gene exists. However, nothing is known about its function. Therefore, we investigated the role of TOR in the GA-producing fungus F. fujikuroi in order to determine whether TOR plays a role in nitrogen regulation, especially in the regulation of GA and bikaverin biosynthesis. We cloned and characterized the F. fujikuroi tor gene. However, we were not able to create knockout mutants, suggesting that TOR is essential for viability. Inhibition of TOR by rapamycin affected the expression of AreA-controlled secondary metabolite genes for GA and bikaverin biosynthesis, as well as genes involved in transcriptional and translational regulation, ribosome biogenesis, and autophagy. Deletion of fpr1 encoding the FKBP12-homologue confirmed that the effects of rapamycin are due to the specific inhibition of TOR. Interestingly, the expression of most of the TOR target genes has been previously shown to be also affected in the glutamine synthetase mutant, although in the opposite way. We demonstrate here for the first time in a filamentous fungus that the TOR kinase is involved in nitrogen regulation of secondary metabolism and that rapamycin affects also the expression of genes involved in translation control, ribosome biogenesis, carbon metabolism, and autophagy.