A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

Alhagi sparsifolia acclimatizes to saline stress by regulating its osmotic, antioxidant, and nitrogen assimilation potential.

BACKGROUND: Alhagi sparsifolia (Camelthorn) is a leguminous shrub species that dominates the Taklimakan desert's salty, hyperarid, and infertile landscapes in northwest China. Although this plant can colonize and spread in very saline soils, how it adapts to saline stress in the seedling stage remains unclear so a pot-based experiment was carried out to evaluate the effects of four different saline stress levels (0, 50, 150, and 300 mM) on the morphological and physio-biochemical responses in A. sparsifolia seedlings. RESULTS: Our results revealed that N-fixing A. sparsifolia has a variety of physio-biochemical anti-saline stress acclimations, including osmotic adjustments, enzymatic mechanisms, and the allocation of metabolic resources. Shoot-root growth and chlorophyll pigments significantly decreased under intermediate and high saline stress. Additionally, increasing levels of saline stress significantly increased Na+ but decreased K+ concentrations in roots and leaves, resulting in a decreased K+/Na+ ratio and leaves accumulated more Na + and K + ions than roots, highlighting their ability to increase cellular osmolarity, favouring water fluxes from soil to leaves. Salt-induced higher lipid peroxidation significantly triggered antioxidant enzymes, both for mass-scavenging (catalase) and cytosolic fine-regulation (superoxide dismutase and peroxidase) of H2O2. Nitrate reductase and glutamine synthetase/glutamate synthase also increased at low and intermediate saline stress levels but decreased under higher stress levels. Soluble proteins and proline rose at all salt levels, whereas soluble sugars increased only at low and medium stress. The results show that when under low-to-intermediate saline stress, seedlings invest more energy in osmotic adjustments but shift their investment towards antioxidant defense mechanisms under high levels of saline stress. CONCLUSIONS: Overall, our results suggest that A. sparsifolia seedlings tolerate low, intermediate, and high salt stress by promoting high antioxidant mechanisms, osmolytes accumulations, and the maintenance of mineral N assimilation. However, a gradual decline in growth with increasing salt levels could be attributed to the diversion of energy from growth to maintain salinity homeostasis and anti-stress oxidative mechanisms.

Performance of Paracoccus pantotrophus MA3 in heterotrophic nitrification-anaerobic denitrification using formic acid as a carbon source.

Excess amount of nitrogen in wastewater has caused serious concerns, such as water eutrophication. Paracoccus pantotrophus MA3, a novel isolated strain of heterotrophic nitrification-anaerobic denitrification bacteria, was evaluated for nitrogen removal using formic acid as the sole carbon source. The results showed that the maximum ammonium removal efficiency was observed under the optimum conditions of 26.25 carbon to nitrogen ratio, 3.39% (v/v) inoculation amount, 34.64 °C temperature, and at 180 rpm shaking speed, respectively. In addition, quantitative real-time PCR technique analysis assured that the gene expression level of formate dehydrogenase, formate tetrahydrofolate ligase, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, respiratory nitrate reductase beta subunit, L-glutamine synthetase, glutamate dehydrogenase, and glutamate synthase were up-regulated compared to the control group, and combined with nitrogen mass balance analysis to conclude that most of the ammonium was removed by assimilation. A small amount of nitrate and nearly no nitrite were accumulated during heterotrophic nitrification. MA3 exhibited significant denitrification potential under anaerobic conditions with a maximum nitrate removal rate of 4.39 mg/L/h, and the only gas produced was N2. Additionally, 11.50 ± 0.06 mg/L/h of NH4+-N removal rate from biogas slurry was achieved.

Nitrogen starvation induces genome-wide activation of transposable elements in Arabidopsis.

Nitrogen (N) availability is a major limiting factor for plant growth and agricultural productivity. Although the gene regulation network in response to N starvation has been extensively studied, it remains unknown whether N starvation has an impact on the activity of transposable elements (TEs). Here, we report that TEs can be transcriptionally activated in Arabidopsis under N starvation conditions. Through genetic screening of idm1-14 suppressors, we cloned GLU1, which encodes a glutamate synthase that catalyzes the synthesis of glutamate in the primary N assimilation pathway. We found that glutamate synthase 1 (GLU1) and its functional homologs GLU2 and glutamate transport 1 (GLT1) are redundantly required for TE silencing, suggesting that N metabolism can regulate TE activity. Transcriptome and methylome analyses revealed that N starvation results in genome-wide TE activation without inducing obvious alteration of DNA methylation. Genetic analysis indicated that N starvation-induced TE activation is also independent of other well-established epigenetic mechanisms, including histone methylation and heterochromatin decondensation. Our results provide new insights into the regulation of TE activity under stressful environments in planta.

Concurrent activation of OsAMT1;2 and OsGOGAT1 in rice leads to enhanced nitrogen use efficiency under nitrogen limitation.

Nitrogen (N) is a major factor for plant development and productivity. However, the application of nitrogenous fertilizers generates environmental and economic problems. To cope with the increasing global food demand, the development of rice varieties with high nitrogen use efficiency (NUE) is indispensable for reducing environmental issues and achieving sustainable agriculture. Here, we report that the concomitant activation of the rice (Oryza sativa) Ammonium transporter 1;2 (OsAMT1;2) and Glutamate synthetase 1 (OsGOGAT1) genes leads to increased tolerance to nitrogen limitation and to better ammonium uptake and N remobilization at the whole plant level. We show that the double activation of OsAMT1;2 and OsGOGAT1 increases plant performance in agriculture, providing better N grain filling without yield penalty under paddy field conditions, as well as better grain yield and N content when plants are grown under N llimitations in field conditions. Combining OsAMT1;2 and OsGOGAT1 activation provides a good breeding strategy for improving plant growth, nitrogen use efficiency and grain productivity, especially under nitrogen limitation, through the enhancement of both nitrogen uptake and assimilation.

New Insights into the Transcriptional Regulation of Genes Involved in the Nitrogen Use Efficiency under Potassium Chlorate in Rice (Oryza sativa L.).

Potassium chlorate (KClO3) has been widely used to evaluate the divergence in nitrogen use efficiency (NUE) between indica and japonica rice subspecies. This study investigated the transcriptional regulation of major genes involved in the NUE in rice treated with KClO3, which acts as an inhibitor of the reducing activity of nitrate reductase (NR) in higher plants. A set of two KClO3 sensitive nitrate reductase (NR) and two nitrate transporter (NRT) introgression rice lines (BC2F7), carrying the indica alleles of NR or NRT, derived from a cross between Saeilmi (japonica, P1) and Milyang23 (indica, P2), were exposed to KClO3 at the seedling stage. The phenotypic responses were recorded 7 days after treatment, and samples for gene expression, physiological, and biochemical analyses were collected at 0 h (control) and 3 h after KClO3 application. The results revealed that Saeilmi (P1, japonica) and Milyang23 (P2, indica) showed distinctive phenotypic responses. In addition, the expression of OsNR2 was differentially regulated between the roots, stem, and leaf tissues, and between introgression lines. When expressed in the roots, OsNR2 was downregulated in all introgression lines. However, in the stem and leaves, OsNR2 was upregulated in the NR introgression lines, but downregulation in the NRT introgression lines. In the same way, the expression patterns of OsNIA1 and OsNIA2 in the roots, stem, and leaves indicated a differential transcriptional regulation by KClO3, with OsNIA2 prevailing over OsNIA1 in the roots. Under the same conditions, the activity of NR was inhibited in the roots and differentially regulated in the stem and leaf tissues. Furthermore, the transcriptional divergence of OsAMT1.3 and OsAMT2.3, OsGLU1 and OsGLU2, between NR and NRT, coupled with the NR activity pattern in the roots, would indicate the prevalence of nitrate (NO3¯) transport over ammonium (NH4+) transport. Moreover, the induction of catalase (CAT) and polyphenol oxidase (PPO) enzyme activities in Saeilmi (P1, KClO3 resistant), and the decrease in Milyang23 (P2, KClO3 sensitive), coupled with the malondialdehyde (MDA) content, indicated the extent of the oxidative stress, and the induction of the adaptive response mechanism, tending to maintain a balanced reduction-oxidation state in response to KClO3. The changes in the chloroplast pigments and proline content propose these compounds as emerging biomarkers for assessing the overall plant health status. These results suggest that the inhibitory potential of KClO3 on the reduction activity of the nitrate reductase (NR), as well as that of the genes encoding the nitrate and ammonium transporters, and glutamate synthase are tissue-specific, which may differentially affect the transport and assimilation of nitrate or ammonium in rice.

Role of Glutamate Synthase in Biofilm Formation by Bacillus subtilis.

Bacillus subtilis forms robust biofilms in the presence of large amounts of carbon sources, such as glycerol. However, little is known about the importance of the metabolic systems, or the relationship between metabolic systems and regulatory systems, involved in biofilm formation. Glutamate synthase, encoded by gltAB, is an enzyme that converts 2-ketoglutarate (a tricarboxylic acid [TCA] cycle intermediate) and glutamine into glutamate, which is a general amino group donor in metabolism. Here, we show that a ΔgltA mutant exhibited early arrest of biofilm formation in complex medium containing glycerol. This phenotype was not due to glutamate auxotrophy. Consistent with its biofilm formation phenotype, the ΔgltA mutant exhibited an early decrease in expression of the epsA and tapA operons, which are responsible for production of biofilm matrix polymers. This resulted from decreased activity of their regulator, Spo0A, as evidenced by reduced expression of other Spo0A-regulated genes in the ΔgltA mutant. The ΔgltA mutation prevented biofilm formation only in the presence of large amounts of glycerol. Moreover, limited expression of citrate synthase (but not other TCA enzymes) restored biofilm-forming ability to the ΔgltA mutant. These results indicate that the ΔgltA mutant accumulates an inhibitory intermediate (citrate) in the TCA cycle in the presence of large amounts of glycerol. The ΔgltA mutant formed biofilms when excess iron was added to the medium. Taken together, the data suggest that accumulation of citrate ions by the ΔgltA mutant causes iron shortage due to chelation, which prevents activation of Spo0A and causes defective biofilm formation.IMPORTANCEBacillus subtilis, a model organism for bacterial biofilm formation, forms robust biofilms in a medium-dependent manner. Although the regulatory network that controls biofilm formation has been well studied, the importance of the underlying metabolic systems remains to be elucidated. The present study demonstrates that a metabolic disorder in a well-conserved metabolic system causes accumulation of an inhibitory metabolic intermediate that prevents activation of the system that regulates biofilm formation. These findings increase our understanding of the coordination between cellular metabolic status and the regulatory networks governing biofilm formation.

Unravelling the lipoyl-relay of exogenous lipoate utilization in Bacillus subtilis.

Lipoate is an essential cofactor for key enzymes of oxidative and one-carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two-step ATP-dependent reaction. Although these pathways were thought to be redundant, a ∆lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl-relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram-positive pathogens.

Glutamate synthase 1 is involved in iron-deficiency response and long-distance transportation in Arabidopsis.

Iron is an essential microelement for plant growth. After uptake from the soil, iron is chelated by ligands and translocated from roots to shoots for subsequent utilization. However, the number of ligands involved in iron chelation is unclear. In this study, we identified and demonstrated that GLU1, which encodes a ferredoxin-dependent glutamate synthase, was involved in iron homeostasis. First, the expression of GLU1 was strongly induced by iron deficiency condition. Second, lesion of GLU1 results in reduced transcription of many iron-deficiency-responsive genes in roots and shoots. The mutant plants revealed a decreased iron concentration in the shoots, and displayed severe leaf chlorosis under the condition of Fe limitation, compared to wild-type. Third, the product of GLU1, glutamate, could chelate iron in vivo and promote iron transportation. Last, we also found that supplementation of glutamate in the medium can alleviate cadmium toxicity in plants. Overall, our results provide evidence that GLU1 is involved in iron homeostasis through affecting glutamate synthesis under iron deficiency conditions in Arabidopsis.

Metabolomics of Escherichia coli Treated with the Antimicrobial Carbon Monoxide-Releasing Molecule CORM-3 Reveals Tricarboxylic Acid Cycle as Major Target.

In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.

Genetic analyses of nitrogen assimilation enzymes in Brassica juncea (L.) Czern & Coss.

Nitrogen (N) is a critical input for plant growth and development. A better understanding of N uptake and utilization is important to develop plant breeding strategies for improving nitrogen use efficiency (NUE). With that objective in mind, we assayed a SNP-genotyped association panel comprising 92 inbred lines for the activities of nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS) and glutamate synthase (GOGAT). All these enzymes are associated with N assimilation. The experiments were carried out at two levels of N application: no added N (N0) and agrnomically recommened dose (100 kg/ha) of N application (N100). Genome wide association studies (GWAS) helped to identify several marker-trait associations (MTAs), involving chromosomes A01, A06, A08, B02, B04, B05 and B08. These explained high phenotypic variation (up to 32%). Annotation of the genomic region(s) in or around significant SNPs allowed prediction of genes encoding high affinity nitrate transporters, glutamine synthetase 1.3, myb-like transcription factor family protein, bidirectional amino acid transporter 1, auxin signaling F-box 3 and oxidoreductases. This is the first attempt to use GWAS for identification of enzyme QTLs to explain variation for nitrogen assimilation enzymes in Brassica juncea.

Molybdenum-Induced Effects on Nitrogen Metabolism Enzymes and Elemental Profile of Winter Wheat (Triticum aestivum L.) Under Different Nitrogen Sources.

Different nitrogen (N) sources have been reported to significantly affect the activities and expressions of N metabolism enzymes and mineral elements concentrations in crop plants. However, molybdenum-induced effects in winter wheat cultivars have still not been investigated under different N sources. Here, a hydroponic study was carried out to investigate these effects on two winter wheat cultivars ('97003' and '97014') as Mo-efficient and Mo-inefficient, respectively, under different N sources (NO3-, NH4NO3, and NH4+). The results revealed that the activities of nitrate reductase (NR) and nitrite reductase (NiR) followed the order of NH4NO3 > NO3- > NH4+ sources, while glutamine synthetase (GS) and glutamate synthase (GOGAT) followed the order of NH4+ > NH4NO3 > NO3- in both the wheat cultivars. However, Mo-induced effects in the activities and expressions of N metabolism enzymes under different N sources followed the order of NH4NO3 > NO3- > NH4+ sources, indicating that Mo has more complementary effects towards nitrate nutrition than the sole ammonium source in winter wheat. Interestingly, under -Mo-deprived conditions, cultivar '97003' recorded more pronounced alterations in Mo-dependent parameters than '97014' cultivar. Moreover, Mo application increased the proteins, amino acids, ammonium, and nitrite contents while concomitantly decreasing the nitrate contents in the same order of NH4NO3 > NO3- > NH4+ sources that coincides with the Mo-induced N enzymes activities and expressions. The findings of the present study indicated that Mo plays a key role in regulating the N metabolism enzymes and assimilatory products under all the three N sources; however, the extent of complementation exists in the order of NH4NO3 > NO3- > NH4+ sources in winter wheat. In addition, it was revealed that mineral elements profiles were mainly affected by different N sources, while Mo application generally had no significant effects on the mineral elements contents in the winter wheat leaves under different N sources.

Glutamate synthases from conifers: gene structure and phylogenetic studies.

BACKGROUND: Plants synthesize glutamate from ammonium by the combined activity of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) through the glutamate synthase cycle. In plants, there are two forms of glutamate synthases that differ in their electron donors, NADH-GOGAT (EC 1.4.1.14) and Fd-GOGAT (EC 1.4.7.1), which have differential roles either in primary ammonia assimilation or in the reassimilation of ammonium from different catabolic processes. Glutamate synthases are complex iron-sulfur flavoproteins containing functional domains involved in the control and coordination of their catalytic activities in annual plants. In conifers, partial cDNA sequences for GOGATs have been isolated and used for gene expression studies. However, knowledge of the gene structure and of phylogenetic relationships with other plant enzymes is quite scant. RESULTS: Technological advances in conifer megagenomes sequencing have made it possible to obtain full-length cDNA sequences encoding Fd- and NADH-GOGAT from maritime pine, as well as BAC clones containing sequences for NADH-GOGAT and Fd-GOGAT genes. In the current study, we studied the genomic organization of pine GOGAT genes, the size of their exons/introns, copy numbers in the pine genome and relationships with other plant genes. Phylogenetic analysis was performed, and the degree of preservation and dissimilarity of key domains for the catalytic activities of these enzymes in different taxa were determined. CONCLUSIONS: Fd- and NADH-GOGAT are encoded by single-copy genes in the maritime pine genome. The Fd-GOGAT gene is extremely large spanning more than 330 kb and the presence of very long introns highlights the important contribution of LTR retrotransposons to the gene size in conifers. In contrast, the structure of the NADH-GOGAT gene is similar to the orthologous genes in angiosperms. Our phylogenetic analysis indicates that these two genes had different origins during plant evolution. The results provide new insights into the structure and molecular evolution of these essential genes.

Biochemical and genetic analyses of N metabolism in maize testcross seedlings: 2. Roots.

KEY MESSAGE: Intracellular factors differentially affected enzyme activities of N assimilation in the roots of maize testcrosses where alanine aminotransferase and glutamate synthase were the main enzymes regulating the levels of glutamate. N is a key macronutrient for plant growth and development. Breeding maize with improved efficiency in N use could help reduce environmental contamination as well as increase profitability for the farmers. Quantitative trait loci (QTL) mapping of traits related to N metabolism in the root tissue was undertaken in a maize testcross mapping population grown in hydroponic cultures. N concentration was negatively correlated with root and total dry mass. Neither the enzyme activities nor metabolites were appreciably correlated between the root and leaf tissues. Repeatability measures for most of the enzymes were lower than for dry mass. Weak negative correlations between most of the enzymes and dry mass resulted likely from dilution and suggested the presence of excess of enzyme activities for maximal biomass production. Glutamate synthase and alanine aminotransferase each explained more variation in glutamate concentration than either aspartate aminotransferase or asparagine synthetase whereas glutamine synthetase was inconsequential. Twenty-six QTL were identified across all traits. QTL models explained 7-43% of the variance with no significant epistasis between the QTL. Thirteen candidate genes were identified underlying QTL within 1-LOD confidence intervals. All the candidate genes were located in trans configuration, unlinked or even on different chromosomes, relative to the known genomic positions of the corresponding structural genes. Our results have implications in improving NUE in maize and other crop plants.

Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum.

Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.

The genome-scale DNA-binding profile of BarR, a ß-alanine responsive transcription factor in the archaeon Sulfolobus acidocaldarius.

BACKGROUND: The Leucine-responsive Regulatory Protein (Lrp) family is a widespread family of regulatory transcription factors in prokaryotes. BarR is an Lrp-like transcription factor in the model archaeon Sulfolobus acidocaldarius that activates the expression of a ß-alanine aminotransferase gene, which is involved in ß-alanine degradation. In contrast to classical Lrp-like transcription factors, BarR is not responsive to any of the α-amino acids but interacts specifically with ß-alanine. Besides the juxtaposed ß-alanine aminotransferase gene, other regulatory targets of BarR have not yet been identified although ß-alanine is the precursor of coenzyme A and thus an important central metabolite. The aim of this study is to extend the knowledge of the DNA-binding characteristics of BarR and of its corresponding regulon from a local to a genome-wide perspective. RESULTS: We characterized the genome-wide binding profile of BarR using chromatin immunoprecipation combined with high-throughput sequencing (ChIP-seq). This revealed 21 genomic binding loci. High-enrichment binding regions were validated to interact with purified BarR protein in vitro using electrophoretic mobility shift assays and almost all targets were also shown to harbour a conserved semi-palindromic binding motif. Only a small subset of enriched genomic sites are located in intergenic regions at a relative short distance to a promoter, and qRT-PCR analysis demonstrated that only one additional operon is under activation of BarR, namely the glutamine synthase operon. The latter is also a target of other Lrp-like transcription factors. Detailed inspection of the BarR ChIP-seq profile at the ß-alanine aminotransferase promoter region in combination with binding motif predictions indicate that the operator structure is more complicated than previously anticipated, consisting of multiple (major and auxiliary) operators. CONCLUSIONS: BarR has a limited regulon, and includes also glutamine synthase genes besides the previously characterized ß-alanine aminotransferase. Regulation of glutamine synthase is suggestive of a link between ß-alanine and α-amino acid metabolism in S. acidocaldarius. Furthermore, this work reveals that the BarR regulon overlaps with that of other Lrp-like regulators.

Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus.

Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase, verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus.

Asparagine Metabolic Pathways in Arabidopsis.

Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages.

Glutamate and histidine improve both solvent yields and the acid tolerance response of Clostridium beijerinckii NCP 260.

AIMS: This study aims to examine the effect of amino acid supplementation on solvent production by Clostridium beijerinckii during the acetone-butanol fermentation and to determine whether amino acids are involved in the acid tolerance response (ATR), which results in increased solvents. METHODS AND RESULTS: Fermentation studies with Cl. beijerinckii NCP 260 in limited-nitrogen media supplemented with glutamate, glutamine, lysine, proline, histidine or asparagine revealed that only glutamate, glutamine or histidine increased butanol titres comparable to control media. Acid survival tests at pH 5 showed that glutamate and histidine were effective in protecting Cl. beijerinckii cells against acid shock, and may be involved in the ATR. Using quantitative PCR, the transcription of the glutamine synthetase, nitrogen regulator and glutamate synthase operon (glnA-nitR-gltAB) was monitored during acid shock conditions, and expression of both the nitR and gltA genes was shown to be increased twofold. CONCLUSIONS: Glutamate and histidine specifically enhance the ATR in Cl. beijerinckii NCP 260, and the genes encoding glutamate synthase and the NitR regulator are both upregulated, predicted to lead to increased endogenous glutamate pools during acidogenesis. This may enhance the ATR and allow more viable cells to enter solventogenesis, thereby increasing butanol titres. Glutamine, glutamate and histidine may also afford protection from butanol stress directly. SIGNIFICANCE AND IMPACT OF THE STUDY: Using substrates naturally rich in glutamine, glutamate and histidine in industrial fermentations is a promising means to increase acid survival and solvent yields in solventogenic Clostridium.

Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective.