Nitrogen Assimilation Pathways Research Articles

Genomic insights into key mechanisms for carbon, nitrogen, and phosphate assimilation by the acidophilic, halotolerant genus Acidihalobacter members.

In-depth comparative genomic analysis was conducted to predict carbon, nitrogen, and phosphate assimilation pathways in the halotolerant, acidophilic genus Acidihalobacter. The study primarily aimed to understand how the metabolic capabilities of each species can determine their roles and effects on the microbial ecology of their unique saline and acidic environments, as well as in their potential application to saline water bioleaching systems. All four genomes encoded the genes for the complete tricarboxylic acid cycle, including 2-oxoglutarate dehydrogenase, a key enzyme absent in obligate chemolithotrophic acidophiles. Genes for a unique carboxysome shell protein, csoS1D, typically found in halotolerant bacteria but not in acidophiles, were identified. All genomes contained lactate and malate utilization genes, but only Ac. ferrooxydans DSM 14175T contained genes for the metabolism of propionate. Genes for phosphate assimilation were present, though organized differently across species. Only Ac. prosperus DSM 5130T and Ac. aeolianus DSM 14174T genomes contained nitrogen fixation genes, while Ac. ferrooxydans DSM 14175T and Ac. yilgarnensis DSM 105917T possessed genes for urease transporters and respiratory nitrate reductases, respectively. The findings suggest that all species can fix carbon dioxide but can also potentially utilize exogenous carbon sources and that the non-nitrogen-fixing species rely on alternative nitrogen assimilation mechanisms.

Response and adaptation of Chlorella pyrenoidosa to 6PPD: Physiological and genetic mechanisms

The extensive contamination of the tire antidegradant N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) in aquatic environments have raised concerns about its potential threats to aquatic organisms. Here, the responses of green algae Chlorella pyrenoidosa (C. pyrenoidosa) to 6PPD exposure were investigated for the first time. The growth of C. pyrenoidosa experienced three sequential phases, including inhibition, recovery and stimulation. Physiological and transcriptome analysis suggested that the growth inhibition was associated with the suppressed nitrogen assimilation and amino acid biosynthesis pathways, among which nitrate transporter (NRT) 2.1 was a key target of 6PPD. Molecular docking revealed the steadily binding of 6PPD to the substrate entry region of NRT 2.1 via hydrogen bonds and π − cation interaction, blocking the acquisition of extracellular inorganic nitrogen. Along with the removal of 6PPD through abiotic processes and biodegradation, an adaptive metabolic shift in cells not only facilitated growth recovery but also triggered a compensatory stimulation phase. With regard to microalgal adaptation, upregulated DNA replication and repair pathways served to maintain the integrity of the genetic information, enhanced photosynthesis cascades and central carbon metabolism improved carbon flux and energy conversion to microalgal biomass, recovered amino acid biosynthesis produced essential proteins for multiple metabolisms. The results provide new insights into microalgal molecular responses to 6PPD exposure, facilitating a better understanding of ecological consequences of 6PPD in the environment.

A dynamic model for predicting biomass and phycocyanin yields in Arthrospira (Spirulina) platensis: A guidance for effective batch cultivation

C-phycocyanin (C-PC) is a highly valuable bioproduct from the cyanobacterium Arthrospira platensis. A crucial factor affecting growth and C-PC production yield is nitrogen nutrients. In this work, an ODE-based dynamic model was constructed to simulate the effect of ammonium concentrations in a batch system on cyanobacterial growth and C-PC production. The model included dynamic regulation of the ammonium transporter and key enzymes involved in the nitrogen assimilation pathway. The prediction of C-PC production, cyanobacterial growth, and remaining ammonium concentration over 24 h strongly correlated with experimental data. Furthermore, the model was able to capture the response of genes involved in ammonium assimilation and C-PC production, as well as the primary metabolites. The dynamic interplay among ammonium, glutamine, and glutamate levels reflects the complexity of nitrogen metabolism in regulating the transcription of genes involved in ammonium uptake, assimilation, and C-PC synthesis and degradation, thus highlighting the cellular response to nitrogen stress. These findings provide a foundation for understanding these biological processes and offer a potential tool for further exploring the complex relationship between nitrogen availability and C-PC accumulation in A. platensis C1 using ammonium as a nitrogen source.

Nitrate assimilation compensates for cell wall biosynthesis in the absence of Aspergillus fumigatus phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.

Uptake of dissolved inorganic nitrogen and N2 fixation by Crocosphaera watsonii under climate change scenarios

The response of N2 fixation to projected future conditions in the ocean cannot be reliably predicted to date. We conducted a minicosm experiment with pre-acclimated cultures of the globally significant diazotroph Crocosphaera watsonii strain WH8501 (“Crocosphaera”). PH and temperature were altered simultaneously to match the RCP scenarios 4.5 and 6 and investigate a more realistic future scenario compared to studies that focus on changes of a single stressor only. The cell abundance and nitrogen metabolism of Crocosphaera was monitored over 5 days. Our results imply that Crocosphaera is able to simultaneously perform N2 fixation and assimilate dissolved inorganic nitrogen (DIN, i.e., nitrate and ammonium) under all the conditions tested and implies a competition with non-diazotrophic phytoplankton for DIN, which should be further investigated. Using NanoSIMS analysis of single cells, our results point towards a preference for DIN assimilation over N2 fixation under more acidic and warmer conditions. Overall, our results show that while the combined alteration of pH and temperature had a negative effect on the diazotroph’s growth and N2 fixation, Crocosphaera is likely to cope well with conditions in the future ocean. The high intra-population variability in nitrogen assimilation pathways may give this species the flexibility to quickly react to environmental changes.

Different nitrogen concentrations affect strawberry seedlings nitrogen form preferences through nitrogen assimilation and metabolic pathways

The preference for different forms of nitrogen (N) is an essential means to improve plant nitrogen utilization efficiency (NUE). Currently, little information is known about the N uptake preferences of strawberries and their interaction with N concentration. In this study, the preference and distribution of nitrate nitrogen (NO3−-N) and ammonium nitrogen (NH4+-N) in strawberry seedlings were investigated in response to different N concentrations. This study used two labeled N sources (15NH4NO3 and NH415NO3) for tracer labeling. Three levels of NH4NO3 were set: low N 5 mM (N1), medium N 15 mM (N2), and high N 25 mM (N3). Each N level included two 15N labeling treatments, totaling six treatments. The results showed that under low N, medium N, and high N conditions, strawberry seedlings exhibited preferences for NH4+-N, NO3−-N, and NH4+-N, respectively. Compared with N1 and N3 treatments, N2 treatment significantly increased the activity of NR, NiR, GS, and GOGAT in strawberry seedlings roots and leaves, and the relative expression levels of FaNRT1.1, FaNRT2.1, and FaAMT1.1 in roots were significantly upregulated; When the N supply concentration increases, the distribution center of NO3−-N in strawberry seedlings shifts from stem to leaf, and under high N conditions, NH4+-N accumulates significantly in the strawberry roots. According our research, the N assimilation and metabolic capacity of strawberries themselves are important factors affecting N form preferences.

Endophytic Bacillus pumilus G5 Interacting with Silicon to Improve Drought Stress Resilience in Glycyrrhiza uralensis Fisch. by Modulating Nitrogen Absorption, Assimilation, and Metabolism Pathways.

Drought stress has become the primary severe threat to global agriculture production, including medicinal plants. Plant growth-promoting bacteria (PGPB) and environmentally friendly element silicon (Si) have emerged as effective methods in alleviating drought stress in various plants. Here, the effects of the plant endophytic G5 interaction with Si on regulating nitrogen absorption, assimilation, and metabolism pathways were investigated in the morphophysiological and gene attributes of Glycyrrhiza uralensis exposed to drought. Results showed that G5+Si application improved nitrogen absorption and assimilation by increasing the available nitrogen content in the soil, further improving the nitrogen utilization efficiency. Then, G5+Si triggered the accumulation of the major adjustment substances proline, γ-aminobutyric acid, putrescine, and chlorophyll, which played an important role in contributing to maintaining balance and energy supply in G. uralensis exposed to drought. These findings will provide new ideas for the combined application of PGPR and Si on both soil and plant systems in a drought habitat.

Revealing the differences in metabolisms and nitrogen removal mechanisms of the full and shortcut nitrification-denitrification processes for the treatment of livestock wastewater

The full nitrification-denitrification process (FN) and shortcut nitrification-denitrification process (SN) are both important approaches for treating livestock wastewater. However, most existing studies focus on pollutant removal performance in wastewater treatment, while the differences between the two processes in terms of bacterial communities, metabolic pathways, nitrogen removal mechanisms, and potential pathogen threats have been poorly investigated. In this study, the SN process exhibited lower oxygen demand, reduced sludge production, higher nitrogen removal rate, and lower pathogen threat for treating livestock wastewater. The bacterial communities, the primary participants of pollutant removal in wastewater treatment, were significantly different between the two processes. In the FN process, 15 genera were significantly enriched, including Saccharimonadales, Micropruina, Ferruginibacter, etc. On the other hand, the SN process showed enrichment of 10 genera, including Brachymonas, Tessaracoccus, Ottowia, etc. These enriched genera, such as Brachymonas, Tessaracoccus, and Ottowia in the SN process, were positively correlated with the denitrification genes, facilitating a higher nitrogen removal rate. Moreover, the FN process exhibited significantly higher amino acid metabolism and reproduction-related pathways, contributing to its higher alpha-diversity of the bacterial community. The significantly enriched genes involved in assimilatory nitrate reduction and nitrogen assimilation pathway suggested that nitrogen assimilation played a crucial role in ammonia nitrogen removal, but it also increased sludge production in the FN process. Additionally, the FN process had a higher number and abundance of potential pathogens, indicating a greater pathogen threat. In conclusion, this study provides valuable insights into the differences between the FN and SN processes in terms of bacterial communities, nitrogen removal mechanisms, and potential pathogen threats. It also offers theoretical guidance for adopting appropriate strategies in wastewater treatment.

Microbial protein production under mixotrophic mode: ammonium assimilation pathway and C/N ratio optimization

Producing microbial protein (MP) is considered as a promising method to alleviate the demand for the global protein supply. The conversion of carbon dioxide (CO2) and organic carbon into MP by hydrogen oxidizing bacteria (HOB) is considered as a potent solution. In this study, under mixotrophic condition, MP production was 2.4 g/L, higher than the sum of that in autotrophic and heterotrophic mode (1.9 g/L), showing the mutualistic relationship of two pathways. According to functional enzyme analysis, the abundance of glutamate dehydrogenase and glutamine synthetase in the heterotrophic and mixotrophic modes was obviously enhanced (80%−800% increment), indicating that adding soluble organics could enhance both the direct (“NH4+ → L-Glutamate”) and the indirect pathway (“NH4+ → L-Glutamine → L-Glutamate”) of NH4+ assimilation in the mixed HOB consortium. For further enhancing MP production, different C/N ratio was evaluated based on mixotrophic mode, achieving amino acid production of 3.3 ± 0.1 g/L and high protein content (58.1%) in C/N-9 reactor. Corynebacterium was dominant (91.4%). In nitrogen assimilation pathway, the higher abundance of glutamine synthetase than glutamate dehydrogenase suggested that NH4+-N was mainly converted via the indirect pathway. With the increase of C/N ratio, 24–48% increment was obtained on glutamine synthetase, along with the decrement of glutamate dehydrogenase. Thus, high C/N ratio could boost the indirect pathway, while performing the negative effect on the direct pathway.

Cell-type proteomic and metabolomic resolution of early and late grain filling stages of wheat endosperm.

The nutritional value of wheat grains, particularly their protein and metabolite composition, is a result of the grain-filling process, especially in the endosperm. Here, we employ laser microdissection (LMD) combined with shotgun proteomics and metabolomics to generate a cell type-specific proteome and metabolome inventory of developing wheat endosperm at the early (15 DAA) and late (26 DAA) grain-filling stages. We identified 1803 proteins and 41 metabolites from four different cell types (aleurone (AL), sub-aleurone (SA), starchy endosperm (SE) and endosperm transfer cells (ETCs). Differentially expressed proteins were detected, 67 in the AL, 31 in the SA, 27 in the SE and 50 in the ETCs between these two-time points. Cell-type accumulation of specific SUT and GLUT transporters, sucrose converting and starch biosynthesis enzymes correlate well with the respective sugar metabolites, suggesting sugar upload and starch accumulation via nucellar projection and ETC at 15 DAA in contrast to the later stage at 26 DAA. Changes in various protein levels between AL, SA and ETC support this metabolic switch from 15 to 26 DAA. The distinct spatial and temporal abundances of proteins and metabolites revealed a contrasting activity of nitrogen assimilation pathways, e.g. for GOGAT, GDH and glutamic acid, in the different cell types from 15 to 26 DAA, which can be correlated with specific protein accumulation in the endosperm. The integration of cell-type specific proteome and metabolome data revealed a complex metabolic interplay of the different cell types and a functional switch during grain development and grain-filling processes.

Characteristics and Mechanism of Heterotrophic Nitrification/Aerobic Denitrification in the Marine Tritonibacter mobilis HN1

This work aimed to reveal the heterotrophic nitrification and aerobic denitrification activities in a salt-tolerant strain, Tritonibacter mobile HN1, isolated from mariculture sludge by using a pure culture method. Strain HN1 showed the removal rates of ammonia, nitrite, nitrate, and total nitrogen of 98.22%, 100%, 95.71%, and 86.63%, respectively, with the carbon source of sodium citrate or sodium succinate, ratio of carbon to nitrogen of 15, salinity of 3%, temperature of 30 °C, shaking speed of 120 rpm, and pH of 8. The genes of narG, narH, narI, nirS, norB, norC, nosZ, glnA, and GltB were found in the genome sequence of strain HN1, confirming the pathways of nitrogen assimilation, heterotrophic nitrification, and aerobic denitrification. In addition, two nitrifying genes, amo and nap, were missing in the genome of Tritonibacter mobile HN1, indicating that HN1 may have novel genes for this function. This study showed that HN1 had the potential to remove nitrogen contaminants in saline environments and was the first Tritonibacter mobilis strain with heterotrophic nitrification and aerobic denitrification capabilities.

Exploring the Application Potential of Aquaculture Sewage Treatment of Pseudomonas chengduensis Strain WD211 Based on Its Complete Genome.

Pseudomonas chengduensis is a new species of Pseudomonas discovered in 2014, and currently, there is a scarcity of research on this bacterium. The P. chengduensis strain WD211 was isolated from a fish pond. This study investigated the purification capability and environmental adaptability of strain WD211 in wastewater and described the basic features and functional genes of its complete genome. According to the results, the sewage treated with strain WD211 showed a decrease in concentration of 18.12% in total nitrogen, 89.39% in NH4+, 62.16% in NO3-, 79.97% in total phosphorus, and 71.41% in COD after 24 h. Strain WD211 is able to survive in a pH range of 6-11. It shows resistance to 7% sodium chloride and different types of antibiotics. Genomic analysis showed that strain WD211 may remove nitrogen and phosphorus through the metabolic pathway of nitrogen assimilation and phosphorus accumulation, and that it can promote organic decomposition through oxygenase. Strain WD211 possesses genes for producing betaine, trehalose, and sodium ion transport, which provide it with salt tolerance. It also has genes for antibiotic efflux and multiple oxidases, which give it antibiotic resistance. This study contributes to the understanding of the sewage treatment ability and potential applications of P. chengduensis.

Glutamate dehydrogenase in "Liverworld"-A study in selected species to explore a key enzyme of plant primary metabolism in Marchantiophyta.

In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts.

Macrogenomic analysis of the effects of aqueous-phase from hydrothermal carbonation of sewage sludge on nitrogen metabolism pathways and associated bacterial communities during composting

The effects of aqueous phases (AP) formed from hydrothermal carbonation of sewage sludge (with or without rice husk) as moisture regulators of nitrogen metabolism pathways during composting are currently unclear. Macrogenomic analyses revealed that both APs resulted in notably changes in bacterial communities during composting; increased levels of nitrogen assimilation, nitrification, and denitrification metabolic pathways; and decreased levels of nitrogen mineralization metabolic pathways. Genes associated with nitrogen assimilation and mineralization accounted for 34–41% and 32–40% of the annotated reads related to nitrogen cycling during composting, respectively, representing them as the most abundant nitrogen metabolism processes. The gudB and norB were identified as key genes for nitrogen mineralization and nitrous oxide emission, respectively. This research offers a better understanding of the effects of additional nitrogen sources on nitrogen metabolism pathways during composting.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrifying-aerobic denitrifying strain H1 as a potential candidate in mariculture wastewater treatment.

The nitrogen removal performance and mechanisms of Bacillus subtilis H1 isolated from a mariculture environment were investigated. Strain H1 efficiently removed NH4+-N, NO2--N, and NO3--N in simulated wastewater with removal efficiencies of 85.61%, 90.58%, and 57.82%, respectively. Strain H1 also efficiently degraded mixed nitrogen (NH4+-N mixed with NO2--N and/or NO3--N) and had removal efficiencies ranging from 82.39 to 89.54%. Nitrogen balance analysis revealed that inorganic nitrogen was degraded by heterotrophic nitrification-aerobic denitrification (HN-AD) and assimilation. 15N isotope tracing indicated that N2O was the product of the HN-AD process, while N2 as the final product was only detected during the reduction of 15NO2--N. The nitrogen assimilation and dissimilation pathways by strain H1 were further clarified using complete genome sequencing, nitrification inhibitor addition, and enzymatic activity measurement, and the ammonium oxidation process was speculated as NH4+ → NH2OH → NO → N2O. These results showed the application prospect of B. subtilis H1 in treating mariculture wastewater.

Characterization of the Nitrogen Removal Potential of Two Newly Isolated Acinetobacter Strains under Low Temperature

Excess nitrogen and phosphorus in the water causes several ecological problems for nutrients. Biological nitrogen removal is an economical and efficient way to prevent excessive nitrogen in the environment. For most areas of China, temperatures are usually lower than 20 °C except during the summertime. It is necessary to discover microbes that can efficiently remove nitrogen at low temperatures. In this study, two Acinetobacter strains were isolated from a sample in a wastewater tank in Taizhou for their capabilities to remove NO3−–N and NO2−–N at 15 °C. Heterotrophic nitrification experiments showed that both strains could efficiently remove nitrogen from the culture medium. The maximum removal rates of NH4+–N were 3.15 mg/L·h and 4.74 mg/L·h for heterotrophic nitrification by the strains F and H, respectively. Strain H grew faster and removed both nitrite and nitrate more efficiently than strain F. Genome sequencing showed that strains F and H could be classified into Acinetobacter johnsonii and Acinetobacter bereziniae, respectively. NO2−–N (100 mg/L) was completely removed in 3 days by strain H. The maximum NO3−–N removal rate was 3.53 mg/L·h for strain F. When strain H was cultured in a broth with 200 mg/L NO3−–N, 97.46% of NH4+–N (200 mg/L) was removed in 5 days, and the maximum NH4+–N removal rate was 4.04 mg/L·h. Genomic sequence analysis showed that both the strains lacked genes involved in the denitrification pathway that transforms NO3− into N2. This implies that nitrate or nitrite is removed through the nitrogen assimilation pathway. Genes responsible for nitrate assimilation are clustered together with molybdopterin cofactor biosynthesis genes. Strain H contains fewer resistance genes and transfer elements. All the above data demonstrate that strain H is a promising candidate for nitrogen removal at lower temperatures. But there is still a lot to be done to systematically evaluate the potential of A. bereziniae strain H in treating wastewater at a pilot scale. These include the long-term performance, environmental tolerance, and nitrogen removal efficiency in wastewater. And the application of these Acinetobacter strains in diverse wastewater treatment settings might require careful optimization and real-time monitoring.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrification–aerobic denitrification bacterium Halomonas sp. DN3

Recently, the functional microorganisms capable of eliminating nitrogenous waste have been applied in mariculture systems. As a potential candidate for treating mariculture wastewater, strain DN3 eliminated 100% of ammonia and nitrate and 96.61%–100% of nitrite within 72 h, when single nitrogen sources at concentrations of 0–50 mg/L. Strain DN3 also exhibited the efficient removal performance of mixed-form nitrogen (ammonia, nitrate, and nitrite) at salinity 30 ‰, C/N ratio 20, and 180 rpm. The nitrogen assimilation pathway dominated inorganic nitrogen metabolism, with less nitrogen (14.23%-25.02% of TN) lost into the air via nitrification and denitrification, based on nitrogen balance analysis. Moreover, the bacterial nitrification pathway was explored by enzymatic assays and inhibition assays. These complex nitrogen assimilation and dissimilation processes were further revealed by bacterial genome analysis. These results provide important insight into nitrogen metabolism of Halomonas sp. and theoretical support for treating mariculture wastewater with strain DN3.

Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy ofRapaza viridis.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here, we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified gene RvNaRL, which had sequence similarity to genes encoding nitrate reductases in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify the function of the protein product RvNaRL, we established RNAi-mediated knock-down and CRISPR-Cas9-mediated knock-out experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knock-down and knock-out cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in the absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains, as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to the acquisition of nitrate assimilation via horizontal gene transfer.

Genome-scale metabolic modeling reveals metabolic trade-offs associated with lipid production in Rhodotorula toruloides.

Rhodotorula toruloides is a non-conventional, oleaginous yeast able to naturally accumulate high amounts of microbial lipids. Constraint-based modeling of R. toruloides has been mainly focused on the comparison of experimentally measured and model predicted growth rates, while the intracellular flux patterns have been analyzed on a rather general level. Hence, the intrinsic metabolic properties of R. toruloides that make lipid synthesis possible are not thoroughly understood. At the same time, the lack of diverse physiological data sets has often been the bottleneck to predict accurate fluxes. In this study, we collected detailed physiology data sets of R. toruloides while growing on glucose, xylose, and acetate as the sole carbon source in chemically defined medium. Regardless of the carbon source, the growth was divided into two phases from which proteomic and lipidomic data were collected. Complemental physiological parameters were collected in these two phases and altogether implemented into metabolic models. Simulated intracellular flux patterns demonstrated the role of phosphoketolase in the generation of acetyl-CoA, one of the main precursors during lipid biosynthesis, while the role of ATP citrate lyase was not confirmed. Metabolic modeling on xylose as a carbon substrate was greatly improved by the detection of chirality of D-arabinitol, which together with D-ribulose were involved in an alternative xylose assimilation pathway. Further, flux patterns pointed to metabolic trade-offs associated with NADPH allocation between nitrogen assimilation and lipid biosynthetic pathways, which was linked to large-scale differences in protein and lipid content. This work includes the first extensive multi-condition analysis of R. toruloides using enzyme-constrained models and quantitative proteomics. Further, more precise kcat values should extend the application of the newly developed enzyme-constrained models that are publicly available for future studies.

15N-labelling of Leaves Combined with GC-MS Analysis as a Tool for Monitoring the Dynamics of Nitrogen Incorporation into Amino Acids.

Labeling plant material such as detached leaves with 15NH4+ is a very instrumental method for the characterization of metabolic pathways of mineral nitrogen assimilation and incorporation into amino acids. A procedure of labeling, followed by amino acid extraction, purification, and derivatization for gas chromatography coupled to mass spectrometry (GC/MS) analysis, is presented. The rationale of heavy isotope abundance calculations and amino acid quantification is detailed. This method is adaptable to various plant species and various kinds of investigations, such as elucidating physiological changes occurring as a result of gene mutations (overexpression or inhibition) in natural variants or genetically modified crops, or characterization of metabolic fluxes in genotypes exhibiting contrasted physiological or developmental adaptive responses to biotic and/or abiotic environmental stresses. Furthermore, the benefit of working on detached organs or pieces of organs is to investigate finely the metabolism of species that are not amenable to laboratory work, such as plants growing in natural environments or under agricultural conditions in the field.