Sulfate Assimilation Pathway Research Articles

Inhibition of sulfur assimilation by S-benzyl-L-cysteine: Impacts on growth, photosynthesis, and leaf proteome of maize plants

Sulfur is an essential nutrient for various physiological processes, including protein synthesis and enzyme activation. We aimed to evaluate how S-benzyl-L-cysteine (SBC), an inhibitor of the sulfur assimilation pathway, affects maize plants' growth, photosynthesis, and leaf proteomic profile. Thus, maize plants were grown for 14 days in vermiculite supplemented with SBC. Photosynthesis was assessed using light and CO2 response curves and chlorophyll a fluorescence. Leaf proteome analysis was conducted to evaluate photosynthetic protein biosynthesis, and ROS content was quantified to assess oxidative stress. Applying SBC resulted in a significant decrease in the growth of maize plants. The gas exchange analysis revealed that maize plants exhibited a diminished rate of CO2 assimilation attributable to both stomatal and non-stomatal limitations. Furthermore, SBC suppressed the activity of important elements involved in the photosynthetic electron transport chain (including photosystems I and II, cytochrome b6f, and ATP synthase) and enzymes responsible for the Calvin cycle, some of which have sulfur-containing prosthetic groups. Consequently, the diminished electron flow rate resulted in a substantial increase in the levels of ROS within the leaves. Our research highlights the crucial role of SBC in disrupting maize photosynthesis by limiting L-cysteine and assimilated sulfur availability, which are essential for the synthesis of protein and prosthetic groups and photosynthetic processes, emphasizing the potential of OAS-TL as a new herbicide site of action.

Enhanced S-adenosyl-L-methionine synthesis in Saccharomyces cerevisiae using metabolic engineering strategies

S-adenosyl-L-methionine (SAM) plays pivotal roles in various physiological processes. With its increasing application in the treatment of diseases such as liver disease, depression, osteoarthritis and Alzheimer's, interest in SAM production aroused. Currently, Saccharomyces cerevisiae is the main industrial producer of SAM. With the surge in demand for SAM, improving the SAM biosynthesis is of importance. In this study, a multimodule engineering strategy was employed to improving SAM production: 1) Enhancing the gene expression of the sulfur assimilation pathway; 2) Strengthening the metabolic flux of the SAM synthesis pathway; 3) Weakening the SAM degradation pathway; 4) Increasing ATP supply. The resulting engineered mutant SC06 (S. cerevisiae CEN.PK2–1CΔgal80∷Tcyc1-sam2-Pgal1-Pgal10-met14-Tadh1, Δlsc2∷Tcyc1-hom6-Pgal1-Pgal10-met6-Tadh1, Δsah1Δmls1) displayed the highest SAM titer of 240.86 mg/L, which was 10.22-fold increase compared with the original strain. With optimized conditions, the SAM titer of mutant SC06 in shake flask fermentation reached 473.02 mg/L with a specific yield of 127.18 mg/g dry cell weight (DCW). In a 5 L fermenter with fed-batch fermentation, the maximal SAM yield of mutant SC06 reached 1.25 g/L with a specific yield of 166.67 mg/g DCW after 58 h cultivation. Therefore, the established metabolic engineering strategies displayed promising efficiency in improving the SAM productivity of S. cerevisiae CEN.PK2–1C, which may provide a useful tool for the improvement of SAM-producing strains.

Functional redundancy of ubiquitin-like sulfur-carrier proteins facilitates flexible, efficient sulfur utilization in the primordial archaeon Thermococcus kodakarensis.

Ubiquitin-like proteins (Ubls) in eukaryotes and bacteria mediate sulfur transfer for the biosynthesis of sulfur-containing biomolecules and form conjugates with specific protein targets to regulate their functions. Here, we investigated the functions and physiological importance of Ubls in a hyperthermophilic archaeon by constructing a series of deletion mutants. We found that the Ubls (TK1065, TK1093, and TK2118) in Thermococcus kodakarensis are conjugated to their specific target proteins, and all three are involved in varying degrees in the biosynthesis of sulfur-containing biomolecules such as tungsten cofactor (Wco) and tRNA thiouridines. TK2118 (named UblB) is involved in the biosynthesis of Wco in a glyceraldehyde 3-phosphate:ferredoxin oxidoreductase, which is required for glycolytic growth, whereas TK1093 (named UblA) plays a key role in the efficient thiolation of tRNAs, which contributes to cellular thermotolerance. Intriguingly, in the presence of elemental sulfur (S0) in the culture medium, defective synthesis of these sulfur-containing molecules in Ubl mutants was restored, indicating that T. kodakarensis can use S0 as an alternative sulfur source without Ubls. Our analysis indicates that the Ubl-mediated sulfur-transfer system in T. kodakarensis is important for efficient sulfur assimilation, especially under low S0 conditions, which may allow this organism to survive in a low sulfur environment.IMPORTANCESulfur is a crucial element in living organisms, occurring in various sulfur-containing biomolecules including iron-sulfur clusters, vitamins, and RNA thionucleosides, as well as the amino acids cysteine and methionine. In archaea, the biosynthesis routes and sulfur donors of sulfur-containing biomolecules are largely unknown. Here, we explored the functions of Ubls in the deep-blanched hyperthermophilic archaeon, Thermococcus kodakarensis. We demonstrated functional redundancy of these proteins in the biosynthesis of tungsten cofactor and tRNA thiouridines and the significance of these sulfur-carrier functions, especially in low sulfur environments. We propose that acquisition of a Ubl sulfur-transfer system, in addition to an ancient inorganic sulfur assimilation pathway, enabled the primordial archaeon to advance into lower-sulfur environments and expand their habitable zone.

Analysis of the Diversity and Functional Potential of Bacterial Communities in Tuberculomas.

The dynamics of lung microbiota in tuberculosis remains poorly understood. Sequencing of variable regions of the 16S rRNA gene from surgically excised tuberculosis foci and biopsy specimens of normal lung tissue allowed characterization of the diversity and predictive potential of bacterial communities. Taxonomic diversity indices attested to differences in the structure of microbial communities between "healthy" lungs and tuberculomas. The microbial composition of "healthy" lungs varied in taxonomic diversity and was presented by both gram-positive and gram-negative bacteria with sufficiently similar metabolic potential. The microbiota of the examined tuberculomas consisted of Mycobacterium tuberculosis in 99.9% of cases. A significant part of the metabolic pathways predicted by PICRUSt2 included cholesterol catabolism, sulfate assimilation, and various pathways for the biosynthesis of cell wall components.

Pungency related gene network in Allium sativum L., response to sulfur treatments

Pungency of garlic (Allium sativum L.) is generated from breakdown of the alk(en)yl cysteine sulphoxide (CSO), alliin and its subsequent breakdown to allicin under the activity of alliinase (All). Based on recent evidence, two other important genes including Sulfite reductase (SiR) and Superoxide dismutase (SOD) are thought to be related to sulfur metabolism. These three gene functions are in sulfate assimilation pathway. However, whether it is involved in stress response in crops is largely unknown. In this research, the order and priority of simultaneous expression of three genes including All, SiR and SOD were measured on some garlic ecotypes of Iran, collected from Zanjan, Hamedan and Gilan, provinces under sulfur concentrations (0, 6, 12, 24 and 60 g/ per experimental unit: pot) using real-time quantitative PCR (RT-qPCR) analysis. For understanding the network interactions between studied genes and other related genes, in silico gene network analysis was constructed to investigate various mechanisms underlying stimulation of A. sativum L. to cope with imposed sulfur. Complicated network including TF-TF, miRNA-TF, and miRNA-TF-gene, was split into sub-networks to have a deeper insight. Analysis of q-RT-PCR data revealed the highest expression in All and SiR genes respectively. To distinguish and select significant pathways in sulfur metabolism, RESNET Plant database of Pathway Studio software v.10 (Elsevier), and other relative data such as chemical reactions, TFs, miRNAs, enzymes, and small molecules were extracted. Complex sub-network exhibited plenty of routes between stress response and sulfate assimilation pathway. Even though Alliinase did not display any connectivity with other stress response genes, it showed binding relation with lectin functional class, as a result of which connected to leucine zipper, exocellulase, peroxidase and ARF functional class indirectly. Integration network of these genes revealed their involvement in various biological processes such as, RNA splicing, stress response, gene silencing by miRNAs, and epigenetic. The findings of this research can be used to extend further research on the garlic metabolic engineering, garlic stress related genes, and also reducing or enhancing the activity of the responsible genes for garlic pungency for health benefits and industry demands.

Complete genome sequence and transcriptome response to vitamin C supplementation of Novacetimonas hansenii SI1 - producer of highly-stretchable cellulose

Novacetimonas hansenii SI1, previously known as Komagataeibacter hansenii, produces bacterial nanocellulose (BNC) with unique ability to stretch. The addition of vitamin C in the culture medium increases the porosity of the membranes and their stretchability making them highly moldable. To better understand the genetic background of this strain, we obtained its complete genome sequence using a hybrid sequencing and assembly strategy. We described the functional regions in the genome which are important for the synthesis of BNC and acetan-like II polymer. We next investigated the effect of 1% vitamin C supplementation on the global gene expression profile using RNA sequencing. Our transcriptomic readouts imply that vitamin C functions mainly as a reducing agent. We found that the changes in cellular redox status are balanced by strong repression of the sulfur assimilation pathway. Moreover, in the reduced conditions, glucose oxidation is decreased and alternative pathways for energy generation, such as acetate accumulation, are activated. The presence of vitamin C negatively influences acetan-like II polymer biosynthesis, which may explain the lowered yield and changed mechanical properties of BNC. The results of this study enrich the functional characteristics of the genomes of the efficient producers of the N. hansenii species. Improved understanding of the adaptation to the presence of vitamin C at the molecular level has important guiding significance for influencing the biosynthesis of BNC and its morphology.

Sulfur assimilation using gaseous carbonyl sulfide by the soil fungus Trichoderma harzianum.

Fungi have the capacity to assimilate a diverse range of both inorganic and organic sulfur compounds. It has been recognized that all sulfur sources taken up by fungi are in soluble forms. In this study, we present evidence that fungi can utilize gaseous carbonyl sulfide (COS) for the assimilation of a sulfur compound. We found that the filamentous fungus Trichoderma harzianum strain THIF08, which has constitutively high COS-degrading activity, was able to grow with COS as the sole sulfur source. Cultivation with 34S-labeled COS revealed that sulfur atom from COS was incorporated into intracellular metabolites such as glutathione and ergothioneine. COS degradation by strain THIF08, in which as much of the moisture derived from the agar medium as possible was removed, indicated that gaseous COS was taken up directly into the cell. Escherichia coli transformed with a COS hydrolase (COSase) gene, which is clade D of the β-class carbonic anhydrase subfamily enzyme with high specificity for COS but low activity for CO2 hydration, showed that the COSase is involved in COS assimilation. Comparison of sulfur metabolites of strain THIF08 revealed a higher relative abundance of reduced sulfur compounds under the COS-supplemented condition than the sulfate-supplemented condition, suggesting that sulfur assimilation is more energetically efficient with COS than with sulfate because there is no redox change of sulfur. Phylogenetic analysis of the genes encoding COSase, which are distributed in a wide range of fungal taxa, suggests that the common ancestor of Ascomycota, Basidiomycota, and Mucoromycota acquired COSase at about 790-670 Ma.IMPORTANCEThe biological assimilation of gaseous CO2 and N2 involves essential processes known as carbon fixation and nitrogen fixation, respectively. In this study, we found that the fungus Trichoderma harzianum strain THIF08 can grow with gaseous carbonyl sulfide (COS), the most abundant and ubiquitous gaseous sulfur compound, as a sulfur source. When the fungus grew in these conditions, COS was assimilated into sulfur metabolites, and the key enzyme of this assimilation process is COS hydrolase (COSase), which specifically degrades COS. Moreover, the pathway was more energy efficient than the typical sulfate assimilation pathway. COSase genes are widely distributed in Ascomycota, Basidiomycota, and Mucoromycota and also occur in some Chytridiomycota, indicating that COS assimilation is widespread in fungi. Phylogenetic analysis of these genes revealed that the acquisition of COSase in filamentous fungi was estimated to have occurred at about 790-670 Ma, around the time that filamentous fungi transitioned to a terrestrial environment.

Challenges and Advances in the Bioproduction of L-Cysteine.

L-cysteine is a proteogenic amino acid with many applications in the pharmaceutical, food, animal feed, and cosmetic industries. Due to safety and environmental issues in extracting L-cysteine from animal hair and feathers, the fermentative production of L-cysteine offers an attractive alternative using renewable feedstocks. Strategies to improve microbial production hosts like Pantoea ananatis, Corynebacterium glutamicum, Pseudomonas sp., and Escherichia coli are summarized. Concerning the metabolic engineering strategies, the overexpression of feedback inhibition-insensitive L-serine O-acetyltransferase and weakening the degradation of L-cysteine through the removal of L-cysteine desulfhydrases are crucial adjustments. The overexpression of L-cysteine exporters is vital to overcome the toxicity caused by intracellular accumulating L-cysteine. In addition, we compiled the process engineering aspects for the bioproduction of L-cysteine. Utilizing the energy-efficient sulfur assimilation pathway via thiosulfate, fermenting cheap carbon sources, designing scalable, fed-batch processes with individual feedings of carbon and sulfur sources, and implementing efficient purification techniques are essential for the fermentative production of L-cysteine on an industrial scale.

A cotton APS REDUCTASE represses root hair elongation via sulfur assimilation and hydrogen peroxide‐mediated cell wall damage

AbstractSulfur is a critical element for plant growth and development, and it is assimilated through a pathway called the sulfur assimilation pathway. APS REDUCTASE (APR) is a key rate‐limiting enzyme in this pathway, and it plays a vital role in regulating sulfur flow and plant development. However, the exact role and mechanism of APR in cotton fiber and root hair development are still not fully understood. This study used in vitro cultured ovules grown on a sulfur‐deficient medium to explore the role of sulfur in fiber development. Genetic approaches such as virus‐induced gene silencing (VIGS) was used to determine the role of target genes. In vitro assays revealed the role of sulfur in fiber development and identified GhAPR2 as a key candidate gene. The GhAPR2 negatively regulates fiber cell initiation and Arabidopsis root hair elongation. Further studies demonstrated that overexpression of GhAPR2 increased the contents of sulfite and GSH to enhance sulfur assimilation in Arabidopsis, leading to abnormal accumulation of ROS in root hair. This down‐regulates the transcription of proline‐rich cell wall protein‐coding genes such as AtEXTs and AtPRPs, reducing the content of hydroxyproline in the cell wall, disrupting the development of the root hair cell wall, and inhibiting root hair elongation. This study elucidated the functional pathway of APR2 in the regulation of root hair development. APR2 first affects sulfur assimilation, which affects the content of reactive oxygen species (ROS), and finally affects the content of hydroxyproline‐rich glycoprotein, which in turn affects the development of root hair. This model also contributes to the understanding of the role of sulfur in fiber development, as well as cell differentiation and development.

QTL mapping reveals novel genes and mechanisms underlying variations in H2S production during alcoholic fermentation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae requirement for reduced sulfur to synthesize methionine and cysteine during alcoholic fermentation, is mainly fulfilled through the sulfur assimilation pathway. Saccharomyces cerevisiae reduces sulfate into sulfur dioxide (SO2) and sulfide (H2S), whose overproduction is a major issue in winemaking, due to its negative impact on wine aroma. The amount of H2S produced is highly strain-specific and also depends on SO2 concentration, often added to grape must. Applying a bulk segregant analysis to a 96-strain-progeny derived from two strains with different abilities to produce H2S, and comparing allelic frequencies along the genome of pools of segregants producing contrasting H2S quantities, we identified two causative regions involved in H2S production in the presence of SO2. A functional genetic analysis allowed the identification of variants in four genes able to impact H2S formation, viz; ZWF1, ZRT2, SNR2, and YLR125W, and involved in functions and pathways not associated with sulfur metabolism until now. These data point out that, in wine fermentation conditions, redox status, and zinc homeostasis are linked to H2S formation while providing new insights into the regulation of H2S production, and a new vision of the interplay between the sulfur assimilation pathway and cell metabolism.

Unraveling the molecular mechanisms of selenite reduction: transcriptomic analysis of Bacillus reveals the key role of sulfur assimilation.

Selenite biotransformation by microorganisms is an effective detoxification and assimilation process. However, current knowledge of the molecular mechanisms of selenite reduction remains circumscribed. Here, the reduction of Se(IV) by a highly selenite-resistant Bacillus sp. SL (up to 50mM) was systematically analyzed, and the molecular mechanisms of selenite reduction were investigated. Remarkably, 10mM selenite was entirely transformed by the strain SL within 20h, demonstrating a faster conversion rate compared to other microorganisms. Furthermore, glutathione (GSH) and exopolysaccharides (EPS) changes were also monitored during the process. Transcriptomic analysis revealed that the genes of ferredoxin-sulfite oxidoreductase (6.82) and sulfate adenylyltransferase (6.32) were significantly upregulated, indicating that the sulfur assimilation pathway is the primary reducing pathway involved in selenite reduction by strain SL. Moreover, key genes associated with NAD(P)/FAD-dependent oxidoreductases and thioredoxin were significantly upregulated. The reduction of Se(IV) was mediated by multiple pathways in strain SL. To our knowledge, this is the initial report to identify the involvement of sulfur assimilation pathway in selenite reduction for bacillus, which is rare in aerobic bacteria.

Inhibition of O-acetylserine (thiol) lyase as a promising new mechanism of action for herbicides

Enzymes of the sulfur assimilation pathway of plants have been identified as potential targets for herbicide development, given their crucial role in synthesizing amino acids, coenzymes, and various sulfated compounds. In this pathway, O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) catalyzes the synthesis of L-cysteine through the incorporation of sulfate into O-acetylserine (OAS). This study used an in silico approach to select seven inhibitors for OAS-TL. The in silico experiments revealed that S-benzyl-L-cysteine (SBC) had a better docking score (−7.0 kcal mol−1) than the substrate OAS (−6.6 kcal mol−1), indicating its suitable interaction with the active site of the enzyme. In vitro experiments showed that SBC is a non-competitive inhibitor of OAS-TL from Arabidopsis thaliana expressed heterologously in Escherichia coli, with a Kic of 4.29 mM and a Kiu of 5.12 mM. When added to the nutrient solution, SBC inhibited the growth of maize and morning glory weed plants due to the reduction of L-cysteine synthesis. Remarkably, morning glory was more sensitive than maize. As proof of its mechanism of action, L-cysteine supplementation to the nutrient solution mitigated the inhibitory effect of SBC on the growth of morning glory. Taken together, our data suggest that reduced L-cysteine synthesis is the primary cause of growth inhibition in maize and morning glory plants exposed to SBC. Furthermore, our findings indicate that inhibiting OAS-TL could potentially be a novel approach for herbicidal action.

Transcription factor OxyR regulates sulfane sulfur removal and L-cysteine biosynthesis in Corynebacterium glutamicum.

Sulfane sulfur, a collective term for hydrogen polysulfide and organic persulfide, often damages cells at high concentrations. Cells can regulate intracellular sulfane sulfur levels through specific mechanisms, but these mechanisms are unclear in Corynebacterium glutamicum. OxyR is a transcription factor capable of sensing oxidative stress and is also responsive to sulfane sulfur. In this study, we found that OxyR functioned directly in regulating sulfane sulfur in C. glutamicum. OxyR binds to the promoter of katA and nrdH and regulates its expression, as revealed via in vitro electrophoretic mobility shift assay analysis, real-time quantitative PCR, and reporting systems. Overexpression of katA and nrdH reduced intracellular sulfane sulfur levels by over 30% and 20% in C. glutamicum, respectively. RNA-sequencing analysis showed that the lack of OxyR downregulated the expression of sulfur assimilation pathway genes and/or sulfur transcription factors, which may reduce the rate of sulfur assimilation. In addition, OxyR also affected the biosynthesis of L-cysteine in C. glutamicum. OxyR overexpression strain Cg-2 accumulated 183 mg/L of L-cysteine, increased by approximately 30% compared with the control (142 mg/L). In summary, OxyR not only regulated sulfane sulfur levels by controlling the expression of katA and nrdH in C. glutamicum but also facilitated the sulfur assimilation and L-cysteine synthesis pathways, providing a potential target for constructing robust cell factories of sulfur-containing amino acids and their derivatives. IMPORTANCE C. glutamicum is an important industrial microorganism used to produce various amino acids. In the production of sulfur-containing amino acids, cells inevitably accumulate a large amount of sulfane sulfur. However, few studies have focused on sulfane sulfur removal in C. glutamicum. In this study, we not only revealed the regulatory mechanism of OxyR on intracellular sulfane sulfur removal but also explored the effects of OxyR on the sulfur assimilation and L-cysteine synthesis pathways in C. glutamicum. This is the first study on the removal of sulfane sulfur in C. glutamicum. These results contribute to the understanding of sulfur regulatory mechanisms and may aid in the future optimization of C. glutamicum for biosynthesis of sulfur-containing amino acids.

The effect of glutathione on glucosinolate biosynthesis through the sulfur assimilation pathway in pakchoi associated with the growth conditions

Glucosinolates (GSLs) are a group of nitrogen- and sulfur-containing secondary metabolites, synthesized primarily in members of the Brassicaceae family, that play an important role in food flavor, plant antimicrobial activity, resistance to insect attack, stress tolerance, and human anti-cancer effects. As a sulfur-containing compound, glutathione has a strong connection with GSLs biosynthesis as a sulfur donor or redox system, and exists in reduced (glutathione; GSH) and oxidized (glutathione disulfide; GSSG) forms. However, the mechanism of GSH regulating GSLs biosynthesis remainds unclear. Hence, the exogenous therapy to pakchoi under normal growth condition and sulfur deficiency condition were conducted in this work to explore the relevant mechanism. The results showed that exogenous application of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased the transcript levels of GSLs synthesis-related genes and transcription factors, as well as sulfur assimilation-related genes under the normal growth condition. Application of exogenous GSH inhibited the expression of GSLs synthesis- and sulfur assimilation-related genes under the normal condition, while the GSLs biosynthesis and the sulfur assimilation pathway were activated by exogenous application of GSH when the content of GSH in vivo of plants decreased owing to sulfur deficiency. Moreover, exogenous application of GSSG increased the transcript levels of GSLs synthesis- and sulfur assimilation-related genes under the normal growth condition and under sulfur deficiency. The present work provides new insights into the molecular mechanisms of GSLs biosynthesis underlying glutathione regulation.

Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus

Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3′-phosphoadenosine 5′-phosphosulfate) released by APS kinase is converted into sulfite and 3′-phosphoadenosine 5′-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was ‘mix-and-matched’ through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.

Adenylyl-Sulfate Kinase (Met14)-Dependent Cysteine and Methionine Biosynthesis Pathways Contribute Distinctively to Pathobiological Processes in Cryptococcus neoformans.

Blocking of nutrient uptake and amino acid biosynthesis are considered potential targets for next-generation antifungal drugs against pathogenic fungi, including Cryptococcus neoformans. In this regard, the sulfate assimilation pathway is particularly attractive, as it is only present in eukaryotes such as plants and fungi, yet not in mammals. Here, we demonstrated that the adenylyl sulfate kinase (Met14) in the sulfate assimilation pathway is not essential yet is required for the viability of C. neoformans due to its involvement in biosynthesis of two sulfur-containing amino acids, cysteine and methionine. Met14-dependent cysteine and methionine biosynthesis was found to significantly contribute to a diverse range of pathobiological processes in C. neoformans. Met14-dependent cysteine rather than methionine biosynthesis was also found to play pivotal roles in cell growth and tolerance to environmental stresses and antifungal drugs. In contrast, the Met14-dependent methionine biosynthesis was found to be more important than cysteine biosynthesis for the production of major cryptococcal virulence factors of melanin pigments and polysaccharide capsules. Finally, we also found that despite its attenuated virulence in an insect model, Galleria mellonella, the met14Δ mutant yielded no difference in virulence in a murine model of systemic cryptococcosis. Hence, clinical inhibition of Met14-dependent amino acid biosynthetic pathways may not be advantageous for the treatment of systemic cryptococcosis. IMPORTANCE Current antifungal drugs have several limitations, such as drug resistance, severe side effects, and a narrow spectrum. Therefore, novel antifungal targets are urgently needed. To this end, fungal sulfur amino acid biosynthetic pathways are considered potential targets for development of new antifungal agents. Here, we demonstrated that Met14 in the sulfate assimilation pathway promotes growth, stress response, and virulence factor production in C. neoformans via synthesis of sulfur-containing amino acids methionine and cysteine. Met14-dependent cysteine rather than methionine synthesis was found to be critical for growth and stress responses, whereas Met14-dependent methionine synthesis was more important for the production of antiphagocytic capsules and antioxidant melanin in C. neoformans. Surprisingly, deletion of the MET14 gene was found to attenuate cryptococcal virulence in an insect model, yet not in a murine model. Collectively, our results showed that Met14-dependent cysteine and methionine biosynthesis play roles that are distinct from each other in C. neoformans. Moreover, Met14 is unlikely to be a suitable anticryptococcal drug target.

New insights into the regulation of plant metabolism by O-acetylserine: sulfate and beyond.

Under conditions of sulfur deprivation, O-acetylserine (OAS) accumulates, which leads to the induction of a common set of six genes, called OAS cluster genes. These genes are induced not only under sulfur deprivation, but also under other conditions where OAS accumulates, such as shift to darkness and stress conditions leading to reactive oxygen species (ROS) or methyl-jasmonate accumulation. Using the OAS cluster genes as a query in ATTED-II, a co-expression network is derived stably spanning several hundred conditions. This allowed us not only to describe the downstream function of the OAS cluster genes but also to score for functions of the members of the co-regulated co-expression network and hence the effects of the OAS signal on the sulfate assimilation pathway and co-regulated pathways. Further, we summarized existing knowledge on the regulation of the OAS cluster and the co-expressed genes. We revealed that the known sulfate deprivation-related transcription factor EIL3/SLIM1 exhibits a prominent role, as most genes are subject to regulation by this transcription factor. The role of other transcription factors in response to OAS awaits further investigation.

SO2 and copper tolerance exhibit an evolutionary trade-off in Saccharomyces cerevisiae.

Copper tolerance and SO2 tolerance are two well-studied phenotypic traits of Saccharomyces cerevisiae. The genetic bases of these traits are the allelic expansion at the CUP1 locus and reciprocal translocation at the SSU1 locus, respectively. Previous work identified a negative association between SO2 and copper tolerance in S. cerevisiae wine yeasts. Here we probe the relationship between SO2 and copper tolerance and show that an increase in CUP1 copy number does not always impart copper tolerance in S. cerevisiae wine yeast. Bulk-segregant QTL analysis was used to identify variance at SSU1 as a causative factor in copper sensitivity, which was verified by reciprocal hemizygosity analysis in a strain carrying 20 copies of CUP1. Transcriptional and proteomic analysis demonstrated that SSU1 over-expression did not suppress CUP1 transcription or constrain protein production and provided evidence that SSU1 over-expression induced sulfur limitation during exposure to copper. Finally, an SSU1 over-expressing strain exhibited increased sensitivity to moderately elevated copper concentrations in sulfur-limited medium, demonstrating that SSU1 over-expression burdens the sulfate assimilation pathway. Over-expression of MET 3/14/16, genes upstream of H2S production in the sulfate assimilation pathway increased the production of SO2 and H2S but did not improve copper sensitivity in an SSU1 over-expressing background. We conclude that copper and SO2 tolerance are conditional traits in S. cerevisiae and provide evidence of the metabolic basis for their mutual exclusivity. These findings suggest an evolutionary driver for the extreme amplification of CUP1 observed in some yeasts.

MicroRNA408 and its encoded peptide regulate sulfur assimilation and arsenic stress response in Arabidopsis.

MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in regulating various developmental and biological processes. The expression of miRNAs is differentially modulated in response to various biotic and abiotic stresses. Recent findings have shown that some pri-miRNAs encode small regulatory peptides known as microRNA-encoded peptides (miPEPs). miPEPs regulate the growth and development of plants by modulating corresponding miRNA expression; however, the role of these peptides under different stress conditions remains unexplored. Here, we report that pri-miR408 encodes a small peptide, miPEP408, that regulates the expression of miR408, its targets, and associated phenotype in Arabidopsis. We also report that miR408, apart from Plantacyanin (ARPN) and Laccase3 (LAC3), targets a glutathione S-transferase (GSTU25) that plays a role in sulfur assimilation and exhibits a range of detoxification activities with the environmental pollutant. Plants overexpressing miR408 showed severe sensitivity under low sulfur (LS), arsenite As(III), and LS + As(III) stress, while miR408 mutants developed using the CRISPR/Cas9 approach showed tolerance. Transgenic lines showed phenotypic alteration and modulation in the expression of genes involved in the sulfur reduction pathway and affect sulfate and glutathione accumulation. Similar to miR408 overexpressing lines, the exogenous application of synthetic miPEP408 and miPEP408OX lines led to sensitivity in plants under LS, As(III), and combined LS + As(III) stress compared to the control. This study suggests the involvement of miR408 and miPEP408 in heavy metal and nutrient deficiency responses through modulation of the sulfur assimilation pathway.

Crystal structure of adenosine 5ʹ-phosphosulfate kinase isolated from Archaeoglobus fulgidus

The 3′-phosphoadenosine-5′-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5′-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.