Nitrogen-limited Growth Conditions Research Articles

Development of a 2-hydroxyglutarate production system by Corynebacterium glutamicum.

2-Oxoglutarate (2-OG) is a tricarboxylate cycle intermediate that can be biologically converted into several industrially important compounds. However, studies on the fermentative production of compounds synthesized from 2-OG, but not via glutamate (defined as 2-OG derivatives), have been limited. Herein, a system that can efficiently produce 2-hydroxyglutarate (2-HG), a 2-OG derivative biosynthesized by the hgdH-encoded NADH-dependent 2-HG dehydrogenase of Acidaminococcus fermentans, was developed as a model using Corynebacterium glutamicum. First, the D3 strain, which lacked the two NADH-consuming enzymes, lactate dehydrogenase and malate dehydrogenase, as well as isocitrate lyase, was constructed as a starting strain. Next, the growth conditions that induced the accumulation of 2-OG were investigated, and it was found that the biotin- and nitrogen-limited (B/N-limited) aerobic growth conditions were suitable for this purpose. Finally, the hgdH gene of A. fermentans became overexpressed in the D3 strain by inserting it into the intergenic regions with the strong constitutive promoter of the tuf gene of C. glutamicum; the engineered strain was cultured under the B/N-limited aerobic growth conditions. The engineered strain produced 80.1mM 2-HG with a yield of 0.390mol/mol glucose, which are the highest titer and yield reported thus far, to the best of our knowledge. Furthermore, reverse genetics showed that the produced 2-HG was partially exported via the YggB protein (NCgl1221 protein, a mechanosensitive channel) known as an exporter for glutamate under the conditions used herein. KEY POINTS: • An efficient 2-HG production system was developed with Corynebacterium glutamicum. • Biotin- and nitrogen-limited aerobic growth conditions induced 2-OG production. • Produced 2-HG was partially excreted via the glutamate exporter, YggB.

Transcriptional Self-Regulation of the Master Nitrogen Regulator GlnR in Mycobacteria.

As a master nitrogen regulator in most actinomycetes, GlnR both governs central nitrogen metabolism and regulates many carbon, phosphate, and secondary metabolic pathways. To date, most studies have been focused on the GlnR regulon, while little is known about the transcriptional regulator for glnR itself. It has been observed that glnR transcription can be upregulated in Mycobacterium smegmatis under nitrogen-limited growth conditions; however, the detailed regulatory mechanism is still unclear. Here, we demonstrate that the glnR gene in M. smegmatis is transcriptionally activated by its product GlnR in response to nitrogen limitation. The precise GlnR binding site was successfully characterized in its promoter region using the electrophoretic mobility shift assay and the DNase I footprinting assay. Site mutagenesis and genetic analyses confirmed that the binding site was essential for in vivo self-activation of glnR transcription. Moreover, based on bioinformatic analyses, we discovered that most of the mycobacterial glnR promoter regions (144 out of 147) contain potential GlnR binding sites, and we subsequently proved that the purified M. smegmatis GlnR protein could specifically bind 16 promoter regions that represent 119 mycobacterial species, including Mycobacterium tuberculosis. Together, our findings not only elucidate the transcriptional self-regulation mechanism of glnR transcription in M. smegmatis but also indicate the ubiquity of the mechanism in other mycobacterial species. IMPORTANCE In actinomycetes, the nitrogen metabolism not only is essential for the construction of life macromolecules but also affects the biosynthesis of secondary metabolites and even virulence (e.g., Mycobacterium tuberculosis). The transcriptional regulation of genes involved in nitrogen metabolism has been thoroughly studied and involves the master nitrogen regulator GlnR. However, the transcriptional regulation of glnR itself remains elusive. Here, we demonstrated that GlnR functions as a transcriptional self-activator in response to nitrogen starvation in the fast-growing model Mycobacterium species Mycobacterium smegmatis. We further showed that this self-regulation mechanism could be widespread in other mycobacteria, which might be beneficial for those slow-growing mycobacteria to adapt to the nitrogen-starvation environments such as within human macrophages for M. tuberculosis.

Non-Proteinogenic Amino Acid β-N-Methylamino-L-Alanine (BMAA): Bioactivity and Ecological Significance.

Research interest in a non-protein amino acid β-N-methylamino-L-alanine (BMAA) arose due to the discovery of a connection between exposure to BMAA and the occurrence of neurodegenerative diseases. Previous reviews on this topic either considered BMAA as a risk factor for neurodegenerative diseases or focused on the problems of detecting BMAA in various environmental samples. Our review is devoted to a wide range of fundamental biological problems related to BMAA, including the molecular mechanisms of biological activity of BMAA and the complex relationships between producers of BMAA and the environment in various natural ecosystems. At the beginning, we briefly recall the most important facts about the producers of BMAA (cyanobacteria, microalgae, and bacteria), the pathways of BMAA biosynthesis, and reliable methods of identification of BMAA. The main distinctive feature of our review is a detailed examination of the molecular mechanisms underlying the toxicity of BMAA to living cells. A brand new aspect, not previously discussed in any reviews, is the effect of BMAA on cyanobacterial cells. These recent studies, conducted using transcriptomics and proteomics, revealed potent regulatory effects of BMAA on the basic metabolism and cell development of these ancient photoautotrophic prokaryotes. Exogenous BMAA strongly influences cell differentiation and primary metabolic processes in cyanobacteria, such as nitrogen fixation, photosynthesis, carbon fixation, and various biosynthetic processes involving 2-oxoglutarate and glutamate. Cyanobacteria were found to be more sensitive to exogenous BMAA under nitrogen-limited growth conditions. We suggest a hypothesis that this toxic diaminoacid can be used by phytoplankton organisms as a possible allelopathic tool for controlling the population of cyanobacterial cells during a period of intense competition for nitrogen and other resources in various ecosystems.

Mycobacterium tuberculosis PknK Substrate Profiling Reveals Essential Transcription Terminator Protein Rho and Two-Component Response Regulators PrrA and MtrA as Novel Targets for Phosphorylation.

ABSTRACTThe Mycobacterium tuberculosis protein kinase K regulates growth adaptation by facilitating mycobacterial survival in response to a variety of in vitro and in vivo stress conditions. Here, we further add that pknK transcription is responsive to carbon and nitrogen starvation signals. The increased survival of an M. tuberculosis ΔpknK mutant strain under carbon- and nitrogen-limiting growth conditions compared to the wild-type (WT) H37Rv suggests an integral role of PknK in regulating growth during metabolic stress. To identify the downstream targets of PknK-mediated signaling, we compared phosphoproteomic and transcription profiles of mycobacterial strains overexpressing WT and phosphorylation-defective PknK. Results implicate PknK as a signaling protein that can regulate several enzymes involved in central metabolism, transcription regulation, and signal transduction. A key finding of this study was the identification of two essential two-component response regulator (RR) proteins, PrrA and MtrA, and Rho transcription terminator, as unique targets for PknK. We confirm that PknK interacts with and phosphorylates PrrA, MtrA, and Rho in vivo. PknK-mediated phosphorylation of MtrA appears to increase binding of the RR to the cognate probe DNA. However, dual phosphorylation of MtrA and PrrA response regulators by PknK and their respective cognate sensor kinases in vitro showed nominal additive effect on the mobility of the protein-DNA complex, suggesting the presence of a potential fine-tuning of the signal transduction pathway which might respond to multiple cues.IMPORTANCE Networks of gene regulation and signaling cascades are fundamental to the pathogenesis of Mycobacterium tuberculosis in adapting to the continuously changing intracellular environment in the host. M. tuberculosis protein kinase K is a transcription regulator that responds to diverse environmental signals and facilitates stress-induced growth adaptation in culture and during infection. This study identifies multiple signaling interactions of PknK and provides evidence that PknK can change the transcriptional landscape during growth transitions by connecting distinctly different signal transduction and regulatory pathways essential for mycobacterial survival.

Identification of species traits enhancing yield in wheat-faba bean intercropping: development and sensitivity analysis of a minimalist mixture model

AimCereal-legume intercropping can result in yield gains compared to monocrops. We aim to identify the combination of crop traits and management practices that confer a yield advantage in strip intercropping.MethodsWe developed a novel, parameter-sparse process-based crop growth model (Minimalist Mixture Model, M3) that can simulate strip intercrops under well-watered but nitrogen limited growth conditions. It was calibrated and validated for spring wheat (Triticum aestivum) and spring faba bean (Vicia faba) grown as monocrops and intercrops, and used to identify the most suitable trait combinations in these intercrops via sensitivity analyses.ResultsThe land equivalent ratio of intercrops was greater than one over a wide range of nitrogen fertilizer levels, but transgressive overyielding, with total yield in the intercrop greater than that of either sole crop, was only obtained at intermediate nitrogen applications. We ranked the local sensitivities of the individual yields of wheat and faba bean of the whole intercrop under various nitrogen input levels to various crop traits.ConclusionsThe total intercrop yield can be improved by selecting specific traits related to phenology of both species, as well as light use efficiency of faba bean and, under high nitrogen applications, of wheat. Changes in height-related crop traits affected individual yields of species in intercrops but not the total intercrop yield.

Rapid, Untargeted Chemical Profiling of Single Cells in Their Native Environment.

We report a method that enables untargeted, high throughput, and quantitative mass spectrometric analysis of single cells from cell suspension without needing additional sample preparation procedures (e.g., molecular tagging) through the combination of single-cell printer technology and liquid vortex capture-mass spectrometry (SCP-LVC-MS). The operating principle behind the SCP-LVC-MS technology is single cell isolation via small droplet piezoelectric ejection followed by capture of the droplet into an LVC-MS sampling probe. Once exposed to an appropriate solvent, the cell is lysed, extracted, and analyzed by MS. The SCP-LVC-MS approach was validated by measuring the lipid composition of microalgae, Chlamydomonas reinhardtii (ChRe) and Euglena gracilis (EuGr), and HeLa cells in their native growth media. Numerous diacylglyceryltrimethylhomo-Ser (DGTS), phosphatidylcholine (PC), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) lipids were observed in single cells. Continuous solvent flow ensures that cells are analyzed rapidly, and no signal carryover between cells is observed. ChRe and EuGr microalgae mixed together in the same solution were differentiated cell-by-cell in real-time based on differences between levels of diacylglyceryltrimethylhomo-Ser (DGTS) and phosphatidylcholine (PC) lipids measured in each cell. Several DGTS lipids present in ChRe were quantified with single-cell resolution by normalizing to a DGTS(32:0) internal standard added to the LVC probe solvent during analysis. Quantitative peak areas were validated by comparing to bulk lipid extracts. Lastly, peak area distributions comprised of hundreds of cells were compared for ChRe after 5 days of nitrogen-limited and normal growth conditions, which show clear differences and the ability to resolve cellular population differences with single-cell resolution.

Bioengineering triacetic acid lactone production in Yarrowia lipolytica for pogostone synthesis.

Yarrowia lipolytica is an oleaginous yeast that is recognized for its ability to accumulate high levels of lipids, which can serve as precursors to biobased fuels and chemicals. Polyketides, such as triacetic acid lactone (TAL), can also serve as a precursor for diverse commodity chemicals. This study used Y. lipolytica as a host organism for the production of TAL via expression of the 2-pyrone synthase gene from Gerbera hybrida. Induction of lipid biosynthesis by nitrogen-limited growth conditions increased TAL titers. We also manipulated basal levels of TAL production using a DNA cut-and-paste transposon to mobilize and integrate multiple copies of the 2-pyrone synthase gene. Strain modifications and batch fermentation in nitrogen-limited medium yielded TAL titers of 2.6 g/L. Furthermore, we show that minimal medium allows TAL to be readily concentrated at >94% purity and converted at 96% yield to pogostone, a valuable antibiotic. Modifications of this reaction scheme yielded diverse related compounds. Thus, oleaginous organisms have the potential to be flexible microbial biofactories capable of economical synthesis of platform chemicals and the generation of industrially relevant molecules.

Dynamics of Photosynthesis in a Glycogen-Deficient glgC Mutant of Synechococcus sp. Strain PCC 7002.

Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting phycobilisomes under nitrogen-limiting growth conditions and secrete a suite of organic acids as a putative reductant-spilling mechanism. This genetic background, therefore, represents an important platform to better understand the complex relationships between light harvesting, photosynthetic electron transport, carbon fixation, and carbon/nitrogen metabolisms. In this study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function of reductant sink manipulation in a glycogen-deficient glgC mutant of Synechococcus sp. strain PCC 7002. The glgC mutant showed increased susceptibility to photoinhibition during the initial phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation, glgC mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild type, the glgC mutant maintained efficient energy transfer from phycobilisomes to photosystem II (PSII) reaction centers, had an elevated PSII/PSI ratio as a result of reduced PSII degradation, and retained a nitrogen-replete-type ultrastructure, including an extensive thylakoid membrane network, after prolonged nitrogen deprivation. Together, these results suggest that multiple global signals for nitrogen deprivation are not activated in the glgC mutant, allowing the maintenance of active photosynthetic complexes under conditions where photosynthesis would normally be abolished.

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440mUmg−1 and 30mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60mM (7.8gl−1), a molar yield of 0.4molmol−1, and a volumetric productivity of 2.1mmoll−1h−1.

First description of small proteins encoded by spRNAs in Methanosarcina mazei strain Gö1.

In Methanosarcina mazei several small RNAs have been identified containing a small putative open reading frame (sORF) and thus classified as spRNAs. Here, we report on the first detection of three small proteins in M.mazei encoded by spRNAs using LC-MS/MS analysis of total protein extracts of cells grown under various stress conditions. Each spRNA shows high conservation in Methanosarcina species with regard to the sORF and the flanking non-coding RNA regions, moreover the predicted RNA structures are as well highly conserved. Characterizing the respective transcript levels in response to several stress conditions by northern blots demonstrated an enormous decrease of spRNA36 and spRNA44 during stationary growth (to less than 5%), and a significant increase of spRNA36 (2.5-fold) in response to nitrogen limitation. spRNA41, however, was only detected by RNA-Seq approaches. Quantification of the small proteins by LC-MS/MS using synthetic stable isotope labeled oligopeptides as standards indicated that the concentration of oligopetide36 and 41 in mid exponential phase is induced under nitrogen limitation, which in case of oligopeptide36 is in accordance with its transcript level. The relative amount of the three oligopeptides did not change upon entering stationary growth phase, even though the transcript levels decreased dramatically. Additional production of the oligopeptides in M.mazei did not result in any evident phenotype under standard or nitrogen limiting growth conditions. However, overall the transcript levels of several genes involved in carbon metabolism or in heat shock response were reduced 2-3 fold due to the overproduction, though no sORF specific change was observed. Based on our findings we hypothesize that oligopeptide36 might have a regulatory function in nitrogen metabolism by modulating the activity of a yet unknown target protein involved in the central nitrogen metabolism.

CcpA-Mediated Catabolite Activation of the Bacillus subtilis ilv-leu Operon and Its Negation by Either CodY- or TnrA-Mediated Negative Regulation

The Bacillus subtilis ilv-leu operon functions in the biosynthesis of branched-chain amino acids. It undergoes catabolite activation involving a promoter-proximal cre which is mediated by the complex of CcpA and P-Ser-HPr. This activation of ilv-leu expression is negatively regulated through CodY binding to a high-affinity site in the promoter region under amino acid-rich growth conditions, and it is negatively regulated through TnrA binding to the TnrA box under nitrogen-limited growth conditions. The CcpA-mediated catabolite activation of ilv-leu required a helix face-dependent interaction of the complex of CcpA and P-Ser-HPr with RNA polymerase and needed a 19-nucleotide region upstream of cre for full activation. DNase I footprinting indicated that CodY binding to the high-affinity site competitively prevented the binding of the complex of CcpA and P-Ser-HPr to cre. This CodY binding not only negated catabolite activation but also likely inhibited transcription initiation from the ilv-leu promoter. The footprinting also indicated that TnrA and the complex of CcpA and P-Ser-HPr simultaneously bound to the TnrA box and the cre site, respectively, which are 112 nucleotides apart; TnrA binding to its box was likely to induce DNA bending. This implied that interaction of TnrA bound to its box with the complex of CcpA and P-Ser-HPr bound to cre might negate catabolite activation, but TnrA bound to its box did not inhibit transcription initiation from the ilv-leu promoter. Moreover, this negation of catabolite activation by TnrA required a 26-nucleotide region downstream of the TnrA box.

Bottom-up Metabolic Reconstruction of Arabidopsis and Its Application to Determining the Metabolic Costs of Enzyme Production.

Large-scale modeling of plant metabolism provides the possibility to compare and contrast different cellular and environmental scenarios with the ultimate aim of identifying the components underlying the respective plant behavior. The existing models of Arabidopsis (Arabidopsis thaliana) are top-down assembled, whereby the starting point is the annotated genome, in particular, the metabolic genes. Hence, dead-end metabolites and blocked reactions can arise that are subsequently addressed by using gap-filling algorithms in combination with species-unspecific genes. Here, we present a bottom-up-assembled, large-scale model that relies solely on Arabidopsis-specific annotations and results in the inclusion of only manually curated reactions. While the existing models are largely condition unspecific by employing a single biomass reaction, we provide three biomass compositions that pertain to realistic and frequently examined scenarios: carbon-limiting, nitrogen-limiting, and optimal growth conditions. The comparative analysis indicates that the proposed Arabidopsis core model exhibits comparable efficiency in carbon utilization and flexibility to the existing network alternatives. Moreover, the model is utilized to quantify the energy demand of amino acid and enzyme de novo synthesis in photoautotrophic growth conditions. Illustrated by the case of the most abundant protein in the world, Rubisco, we determine its synthesis cost in terms of ATP requirements. This, in turn, allows us to explore the tradeoff between protein synthesis and growth in Arabidopsis. Altogether, the model provides a solid basis for completely species-specific integration of high-throughput data, such as gene expression levels, and for condition-specific investigations of in silico metabolic engineering strategies.

The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. Δppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.

Concerted Changes in Gene Expression and Cell Physiology of the CyanobacteriumSynechocystissp. Strain PCC 6803 during Transitions between Nitrogen and Light-Limited Growth

Physiological adaptation and genome-wide expression profiles of the cyanobacterium Synechocystis sp. strain PCC 6803 in response to gradual transitions between nitrogen-limited and light-limited growth conditions were measured in continuous cultures. Transitions induced changes in pigment composition, light absorption coefficient, photosynthetic electron transport, and specific growth rate. Physiological changes were accompanied by reproducible changes in the expression of several hundred open reading frames, genes with functions in photosynthesis and respiration, carbon and nitrogen assimilation, protein synthesis, phosphorus metabolism, and overall regulation of cell function and proliferation. Cluster analysis of the nearly 1,600 regulated open reading frames identified eight clusters, each showing a different temporal response during the transitions. Two large clusters mirrored each other. One cluster included genes involved in photosynthesis, which were up-regulated during light-limited growth but down-regulated during nitrogen-limited growth. Conversely, genes in the other cluster were down-regulated during light-limited growth but up-regulated during nitrogen-limited growth; this cluster included several genes involved in nitrogen uptake and assimilation. These results demonstrate complementary regulation of gene expression for two major metabolic activities of cyanobacteria. Comparison with batch-culture experiments revealed interesting differences in gene expression between batch and continuous culture and illustrates that continuous-culture experiments can pick up subtle changes in cell physiology and gene expression.

Variación cuantitativa y cualitativa de la composición en ácidos grasos de <i>Crypthecodinium cohnii</i> en condiciones de supresión de nitrógeno

Crypthecodinium cohnii is a heterotrophic dinoflagellate that can achieve a lipid content greater than 20% on dry weight. DHA can represent up to 40% of the total fatty acids. Our objective was to investigate the variation in fatty acid content when growth conditions are optimal compared with when there is limited growth, due to the absence of nitrogen in the media. Nitrogen-limited growth conditions caused the cells to stop dividing and accumulate lipids, principally as docohexanoic acid (DHA). Oxygen availability in the culture favoured DHA accumulation. In the cultures without nitrogen and with oxygen availability there was a lipid cell content 3,18 times higher than in the control condition (with nitrogen and air), and 2,25 higher than those without nitrogen and air. C. cohnii can be used as an optimal DHA source for the production of phytodiets in aquaculture.

Improvement of the conversion of polystyrene to polyhydroxyalkanoate through the manipulation of the microbial aspect of the process: A nitrogen feeding strategy for bacterial cells in a stirred tank reactor

Pseudomonas putida CA-3 has been shown to accumulate the biodegradable plastic polyhydroxyalkanoate (PHA) when fed styrene or polystyrene pyrolysis oil as the sole carbon and energy source under nitrogen limiting growth conditions (67 mg nitrogen per litre at time 0). Batch fermentation of P. putida CA-3 grown on styrene or polystyrene pyrolysis oil in a stirred tank reactor yields PHA at 30% of the cell dry weight (CDW). The feeding of nitrogen at a rate of 1 mg N/l/h resulted in a 1.1-fold increase in the percentage of CDW accumulated as PHA. An increase in the rate of nitrogen feeding up to 1.5 mg N/l/h resulted in further increases in the percentage of the cell dry weight composed of PHA. However, feeding rates of 1.75 and 2 mg N/l/h resulted in dramatic decreases in the percentage of cell dry weight composed of PHA. Interestingly nitrogen was not detectable in the growth medium after 16 h, in any of the growth conditions tested. A higher cell density was observed in cells supplied with nitrogen and thus further increases in the overall production of PHA were observed through nitrogen feeding. The highest yield of PHA was 0.28 g PHA per g styrene supplied with a nitrogen feeding rate of 1.5 mg/l/h.

Production of itaconic acid by Pseudozyma antarctica NRRL Y-7808 under nitrogen-limited growth conditions

Pseudozyma antarctica NRRL Y-7808 was found to produce itaconic acid from glucose and other sugars under nitrogen-limited growth conditions. Other Pseudozyma strains screened, including a second strain of P. antarctica, did not produce this product; therefore itaconic acid production is not a common trait of the genus. Glucose and fructose were the most efficiently utilized substrates, followed by sucrose and maltose; lactose and glycerol were the poorest substrates. The maximum yield in flask experiments was 37.5% (approximately 30 g/L itaconic acid from 80 g/L glucose). The maximum rate of production in flask cultures was 248 mg L −1 h −1 at a C/N ratio of 116, while the best combination of rate and yield was produced at a C/N ratio of 175 (230 mg L −1 h −1 and 29%, respectively). A stirred-tank reactor process study resulted in a 20% yield and volumetric production rate of 110 mg L −1 h −1.

Autophagic Nutrient Recycling in Arabidopsis Directed by the ATG8 and ATG12 Conjugation Pathways

Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogen-limiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.

Isotopic estimates of new carbon inputs into litter and soils in a four-year climate change experiment with Douglas-fir

Because soil is a major reservoir of terrestrial carbon and a potential sink for atmospheric CO2, determining plant inputs to soil carbon is critical for understanding ecosystem carbon dynamics. We present a modified method to quantify the effects of global climate change on plant inputs of carbon to soil based on 13C:12C ratio (δ13C) analyses that accounts for isotopic fractionation between inputs and newly created soil carbon. In a four-year study, the effects of elevated CO2 and temperature were determined for reconstructed Douglas-fir (Pseudotsuga mensiezii (Mirb.) Franco) ecosystems in which native soil of low nitrogen content was used. The δ13C patterns in litter and mineral soil horizons were measured and compared to δ13C patterns in live needles, fine roots, and coarse roots. From regression analyses, we calculated the isotopic enrichment in 13C of newly incorporated soil carbon relative to needle and root carbon at 4‰ and 2‰, respectively. These enrichments must be considered when using shifts in soil δ13C to calculate inputs of plant carbon into the soil, and are probably a major factor in the progressive enrichment in 13C with increasing depth in soil profiles. Relative to the total carbon in each layer, the proportion of new carbon from recent photosynthate in each soil layer was 13–15% in the A horizon, 7–9% in litter layers, and 4% in the B2 and C horizons. New carbon in the A horizon was estimated at 370 g C m−2. Carbon concentrations and new carbon in A horizons were correlated (r 2=0.78, n=12), but with a slope of 0.356, indicating that about 36% of net carbon accumulation in the A horizon was from inputs via roots, root exudates or mycorrhizal fungi and 64% of carbon was derived from surface litter decomposition. Under the nitrogen-limited growth conditions used in this study, neither elevated CO2 nor temperature affected soil carbon sequestration patterns.

The nitrogen assimilation control (Nac) protein represses asnC and asnA transcription in Escherichia coli

In this work, we show that the expression of the asnA and asnC genes is regulated by the availability of ammonium in the growth medium. Our results suggest that, under nitrogen-limiting growth conditions, the nitrogen assimilation control (Nac) protein is involved in the repression of the asnC gene, whose product is required to activate the transcription of asnA. We also show that asparagine negatively affects the expression of asnA, independently of the presence of Nac. These results allow us to conclude that asnA transcription is regulated by two different mechanisms that respond to different effectors: nitrogen and asparagine availability.