SLC26A1 is a major determinant of sulfate homeostasis in humans.

Sulfate plays a pivotal role in numerous physiological processes in the human body, including bone and cartilage health. A role of the anion transporter SLC26A1 (Sat1) for sulfate reabsorption in the kidney is supported by the observation of hyposulfatemia and hypersulfaturia in Slc26a1-knockout mice. The impact of SLC26A1 on sulfate homeostasis in humans remains to be defined. By combining clinical genetics, functional expression assays, and population exome analysis, we identify SLC26A1 as a sulfate transporter in humans and experimentally validate several loss-of-function alleles. Whole-exome sequencing from a patient presenting with painful perichondritis, hyposulfatemia, and renal sulfate wasting revealed a homozygous mutation in SLC26A1, which has not been previously described to the best of our knowledge. Whole-exome data analysis of more than 5,000 individuals confirmed that rare, putatively damaging SCL26A1 variants were significantly associated with lower plasma sulfate at the population level. Functional expression assays confirmed a substantial reduction in sulfate transport for the SLC26A1 mutation of our patient, which we consider to be novel, as well as for the additional variants detected in the population study. In conclusion, combined evidence from 3 complementary approaches supports SLC26A1 activity as a major determinant of sulfate homeostasis in humans. In view of recent evidence linking sulfate homeostasis with back pain and intervertebral disc disorder, our study identifies SLC26A1 as a potential target for modulation of musculoskeletal health.

Genome-wide identification, structural and gene expression analysis of the nitrate transporters (NRTs) family in potato (Solanum tuberosum L.).

Nitrogen (N2) is the most important source of mineral N for plant growth, which was mainly transported by nitrate transporters (NRTs). However, little is known about the NRT gene family in potato. In this study, StNRT gene family members were identified in potato. In addition, we performed StNRT subfamily classification, gene structure and distribution analysis, and conserved domain prediction using various bioinformatics tools. Totally, 39 StNRT gene members were identified in potato genome, including 33, 4 and 2 member belong to NRT1, NRT2, and NRT3, respectively. These 39 StNRT genes were randomly distributed on all chromosomes. The collinearity results show that StNRT members in potato are closely related to Solanum lycopersicum and Solanum melongena. For the expression, different members of StNRT play different roles in leaves and roots. Especially under sufficient nitrogen conditions, different members have a clear distribution in different tissues. These results provide valuable information for identifying the members of the StNRT family in potato and could provide functional characterization of StNRT genes in further research.

Ameliorative effect and mechanism of Yi-Suan-Cha against hyperuricemia in rats.

BACKGROUND: This study aimed to evaluate the urate-lowering effects of Yi-Suan-Cha and explore its underlying mechanisms in experimental hyperuricemia induced in rats. METHODS: Forty-eight male SD rats were randomly allocated into normal control, model, allopurinol, benzbromarone, low-dose Yi-Suan-Cha (0.2 g/ml), and high-dose Yi-Suan-Cha (0.4 g/ml) groups (n = 8 rats per group). Rat models of hyperuricemia were established through intragastric administration of adenine 25 mg/kg + potassium oxalate 300 mg/kg for 3 weeks. After the last administration, serum uric acid, creatinine, and urea nitrogen levels were measured. Renal histopathology was observed by hematoxylin-eosin staining. Xanthine oxidase level in serum and liver homogenates was measured by ELISA. The protein and mRNA expression of URAT1, ABCG2, OAT1, and GLUT9 in the kidney was detected by Western blotting and RT-PCR, respectively. RESULTS: The serum uric acid levels were significantly lowered in all medication groups than in the model group. The benzbromarone and both Yi-Suan-Cha groups showed clear kidney structures with no obvious abnormalities. Compared with the normal control group, the model group showed increased URAT1/GLUT9 protein expression and decreased ABCG2/OAT1 protein expression. Compared with the model group, both Yi-Suan-Cha groups showed decreased URAT1/GLUT9 protein expression and increased ABCG2/OAT1 protein expression. Compared with that in the normal control group, URAT1/GLUT9 mRNA expression increased in the model group. Compared with the model group, the low-dose and high-dose Yi-Suan-Cha groups showed decreased URAT1/GLUT9 mRNA expression and increased ABCG2/OAT1 mRNA expression. CONCLUSION: Yi-Suan-Cha may lower uric acid level by downregulating URAT1/GLUT9 expression and upregulating ABCG2/OAT1 expression.

Arabidopsis NRT1.2 interacts with the PHOSPHOLIPASE Dα1 (PLDα1) to positively regulate seed germination and seedling development in response to ABA treatment.

NRT1.2 has been characterized as a low-affinity nitrate transporter and an abscisic acid (ABA) transporter in Arabidopsis. In this study, we demonstrate that NRT1.2 positively regulated the ABA response during germination and seedling development. The transgenic Arabidopsis NRT1.2-over-expressionors showed increased sensitivity to ABA during these processes. qRT-PCR assays indicated that NRT1.2 over-production in 7-days-old seedlings up-regulated the expression of ABA-responsive genes: ABI1, ABI2, ABI3, ABI4, ABI5, RAB18, RD29A, and RD29B and PHOSPHOLIPASE Dα1 (PLDα1). The expression of these genes was suppressed in the nrt1.2 mutant in comparison with the wild type following ABA treatment. Importantly, bimolecular fluorescence complementation assays indicated that NRT1.2 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by using yeast two hybrid (Y2H) experiments with the mating-based split ubiquitin system (MbSUS). Moreover, genetic assays indicated that PLDα1 acts epistatically on NRT1.2 to affect ABA signaling. Taken together, our results provide detailed mechanisms of NRT1.2 in ABA-mediated seed germination and seedling development.

Genome-Wide Systematic Characterization of the NPF Family Genes and Their Transcriptional Responses to Multiple Nutrient Stresses in Allotetraploid Rapeseed.

NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) family (NPF) proteins can transport various substrates, and play crucial roles in governing plant nitrogen (N) uptake and distribution. However, little is known about the NPF genes in Brassica napus. Here, a comprehensive genome-wide systematic characterization of the NPF family led to the identification of 193 NPF genes in the whole genome of B. napus. The BnaNPF family exhibited high levels of genetic diversity among sub-families but this was conserved within each subfamily. Whole-genome duplication and segmental duplication played a major role in BnaNPF evolution. The expression analysis indicated that a broad range of expression patterns for individual gene occurred in response to multiple nutrient stresses, including N, phosphorus (P) and potassium (K) deficiencies, as well as ammonium toxicity. Furthermore, 10 core BnaNPF genes in response to N stress were identified. These genes contained 6-13 transmembrane domains, located in plasma membrane, that respond discrepantly to N deficiency in different tissues. Robust cis-regulatory elements were identified within the promoter regions of the core genes. Taken together, our results suggest that BnaNPFs are versatile transporters that might evolve new functions in B. napus. Our findings benefit future research on this gene family.

The Effect of Substituted Benzene-Sulfonamides and Clinically Licensed Drugs on the Catalytic Activity of CynT2, a Carbonic Anhydrase Crucial for Escherichia coli Life Cycle.

Proteins are relevant antimicrobial drug targets, and among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central metabolism, or are necessary for the pathogen vitality. Genomic exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant ß-CA (CynT2) identified in the genome of the Gram-negative bacterium Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO2. Surprisingly, this enzyme has not been investigated for its inhibition with any class of CA inhibitors. Here, we show that CynT2 was strongly inhibited by some substituted benzene-sulfonamides and the clinically used inhibitor sulpiride (KIs in the range of 82-97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure-activity relationship for this class of enzyme inhibitors.

Identification of Molecular Integrators Shows that Nitrogen Actively Controls the Phosphate Starvation Response in Plants.

Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other's transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.

SULTR3s Function in Chloroplast Sulfate Uptake and Affect ABA Biosynthesis and the Stress Response.

Plants are major sulfur reducers in the global sulfur cycle. Sulfate, the major natural sulfur source in soil, is absorbed by plant roots and transported into plastids, where it is reduced and assimilated into Cys for further metabolic processes. Despite its importance, how sulfate is transported into plastids is poorly understood. We previously demonstrated using single Arabidopsis (Arabidopsis thaliana) genetic mutants that each member of the sulfate transporter (SULTR) subfamily 3 was able to transport sulfate across the chloroplast envelope membrane. To resolve the function of SULTR3s, we constructed a sultr3 quintuple mutant completely knocking out all five members of the subfamily. Here we report that all members of the SULTR3 subfamily show chloroplast membrane localization. Sulfate uptake by chloroplasts of the quintuple mutant is reduced by more than 50% compared with the wild type. Consequently, Cys and abscisic acid (ABA) content are reduced to ∼67 and ∼20% of the wild-type level, respectively, and strong positive correlations are found among sulfate, Cys, and ABA content. The sultr3 quintuple mutant shows obvious growth retardation with smaller rosettes and shorter roots. Seed germination of the sultr3 quintuple mutant is hypersensitive to exogenous ABA and salt stress, but is rescued by sulfide supplementation. Furthermore, sulfate-induced stomatal closure is abolished in the quintuple mutant, strongly suggesting that chloroplast sulfate is required for stomatal closure. Our genetic analyses unequivocally demonstrate that sulfate transporter subfamily 3 is responsible for more than half of the chloroplast sulfate uptake and influences downstream sulfate assimilation and ABA biosynthesis.

Mitochondrial Pyruvate Carriers Prevent Cadmium Toxicity by Sustaining the TCA Cycle and Glutathione Synthesis.

Cadmium (Cd) is a major heavy metal pollutant, and Cd toxicity is a serious cause of abiotic stress in the environment. Plants protect themselves against Cd stress through a variety of pathways. In a recent study, we found that mitochondrial pyruvate carriers (MPCs) are involved in Cd tolerance in Arabidopsis (Arabidopsis thaliana). Following the identification of MPCs in yeast (Saccharomyces cerevisiae) in 2012, most studies have focused on the function of MPCs in animals, as a possible approach to reduce the risk of cancer developing. The results of this study show that AtMPC protein complexes are required for Cd tolerance and prevention of Cd accumulation in Arabidopsis. AtMPC complexes are composed of two elements, AtMPC1 and AtMPC2 (AtNRGA1 or AtMPC3). When the formation of AtMPCs was interrupted by the loss of AtMPC1, glutamate could supplement the synthesis of acetyl-coenzyme A and sustain the TCA cycle. With the up-regulation of glutathione synthesis following exposure to Cd stress, the supplementary pathway could not efficiently drive the tricarboxylic acid cycle without AtMPC. The ATP content decreased concomitantly with the deletion of tricarboxylic acid activity, which led to Cd accumulation in Arabidopsis. More importantly, ScMPCs were also required for Cd tolerance in yeast. Our results suggest that the mechanism of Cd tolerance may be similar in other species.

NRT1.1B improves selenium concentrations in rice grains by facilitating selenomethinone translocation.

Selenium (Se) is an essential trace element for humans and other animals, yet approximately one billion people worldwide suffer from Se deficiency. Rice is a staple food for over half of the world's population that is a major dietary source of Se. In paddy soils, rice roots mainly take up selenite. Se speciation analysis indicated that most of the selenite absorbed by rice is predominantly transformed into selenomethinone (SeMet) and retained in roots. However, the mechanism by which SeMet is transported in plants remains largely unknown. In this study, SeMet uptake was found to be an energy-dependent symport process involving H+ transport, with neutral amino acids strongly inhibiting SeMet uptake. We further revealed that NRT1.1B, a member of rice peptide transporter (PTR) family which plays an important role in nitrate uptake and transport in rice, displays SeMet transport activity in yeast and Xenopus oocyte. The uptake rate of SeMet in the roots and its accumulation rate in the shoots of nrt1.1b mutant were significantly repressed. Conversely, the overexpression of NRT1.1B in rice significantly promoted SeMet translocation from roots to shoots, resulting in increased Se concentrations in shoots and rice grains. With vascular-specific expression of NRT1.1B, the grain Se concentration was 1.83-fold higher than that of wild type. These results strongly demonstrate that NRT1.1B holds great potential for the improvement of Se concentrations in grains by facilitating SeMet translocation, and the findings provide novel insight into breeding of Se-enriched rice varieties.

Research on the pharmacodynamics and mechanism of Fraxini Cortex on hyperuricemia based on the regulation of URAT1 and GLUT9.

Fraxini Cortex (also known as Qinpi, QP) has been used for the treatment of hyperuricemia with a significant difference on efficacy of QP from different regions. However, it`s still unknown whether proportion of components is the key and why same kind of herbs have different therapeutic effects. In this study, different sources of QP were collected from Shaanxi Qinpi extracts (SQPE), Henan Qinpi extracts (HQPE), Hebei Qinpi extracts (GQPE) provinces in China. Rat model of hyperuricemia with hypoxanthine combined with potassium oxonate were established to determine the levels of blood urea nitrogen (BUN), serum uric acid (SUA), urine uric acid (UUA) and creatinine (Cr). Hematoxylin-eosin staining (H&E) and Periodic Acid-Schiff staining (PAS) were performed for renal pathology while Western blot analysis and real-time PCR analysis for proteins and mRNA expression levels. High-performance liquid chromatograph (HPLC) was used for components and composition analysis. Our results demonstrated that QPE from different regions could alleviate hyperuricemia via increasing significantly the SCr and BUN levels whereas decreasing markedly UCr, SUA and UUA levels. Additionally, QPE could also improve the pathological changes of the kidneys. The protein and mRNA levels of urate reabsorption transporter 1 (URAT1) and glucose transporter 9 (GLUT9) were down-regulated by QPE treatment. SQPE hold a better activity on improving hyperuricemia and regulating URAT1 and GLUT9. HPLC analysis showed that the proportion of four components aesculin, aesculetin, fraxin, fraxetin were 9.002: 0.350: 8.980: 0.154 (SQPE); 0.526: 0.164: 7.938: 0.102 (HQPE); 12.022: 1.65: 0.878: 1.064 (GQPE). These data indicate that this proportion of effective components may be an important factor for efficacy of QP and had implications for the treatment of hyperuricemia.

A NIGT1-centred transcriptional cascade regulates nitrate signalling and incorporates phosphorus starvation signals in Arabidopsis.

Nitrate is a nutrient signal that triggers complex regulation of transcriptional networks to modulate nutrient-dependent growth and development in plants. This includes time- and nitrate concentration-dependent regulation of nitrate-related gene expression. However, the underlying mechanisms remain poorly understood. Here we identify NIGT1 transcriptional repressors as negative regulators of the Arabidopsis NRT2.1 nitrate transporter gene, and show antagonistic regulation by NLP primary transcription factors for nitrate signalling and the NLP-NIGT1 transcriptional cascade-mediated repression. This antagonistic regulation provides a resolution to the complexity of nitrate-induced transcriptional regulations. Genome-wide analysis reveals that this mechanism is applicable to NRT2.1 and other genes involved in nitrate assimilation, hormone biosynthesis and transcription. Furthermore, the PHR1 master regulator of the phosphorus-starvation response also directly promotes expression of NIGT1 family genes, leading to reductions in nitrate uptake. NIGT1 repressors thus act in two transcriptional cascades, forming a direct link between phosphorus and nitrogen nutritional regulation.

Overexpression of the nitrate transporter, OsNRT2.3b, improves rice phosphorus uptake and translocation.

KEY MESSAGE: Overexpression of OsNRT2.3b in rice can increase Pi uptake and accumulation through advanced root system, enhanced OsPT and OsPHR genes expression, and the phloem pH homeostasis. Nitrogen (N) and phosphorus (P) are two essential macronutrients for plants. Overexpression of the rice nitrate transporter, OsNRT2.3b, can improve rice grain yield and nitrogen use efficiency (NUE). Here, OsNRT2.3b overexpression resulted in increased grain yield, straw yield, and grain:straw ratio, accompanied by increased P concentrations in the leaf blade, leaf sheath, culm, and unfilled rice hulls. Overexpression of OsNRT2.3b significantly increased 33Pi uptake compared with WT under 300-µM Pi but not 10-µM Pi condition in 24 h. Moreover, the OsNRT2.3b-overexpressing rice lines showed increased root and shoot biomass, root:shoot ratio, total root length root surface area and N, P accumulation under 300- and 10-µM Pi supply in hydroponic solution. The levels of OsPT2, OsPT8, and OsPHR2 expression in roots and of OsPT1 and OsPHR2 in shoots were upregulated in OsNRT2.3b-overexpressing rice. These results indicated that OsNRT2.3b overexpression can improve rice P uptake and accumulation, partially through the advanced root system, enhanced gene expression, and the phloem pH regulation function.

A New Genetic Diagnostic for Enlarged Vestibular Aqueduct Based on Next-Generation Sequencing.

Enlarged vestibular aqueduct (EVA) is one of the most common congenital inner ear malformations and accounts for 1-12% of sensorineural deafness in children and adolescents. Multiple genetic defects contribute to EVA; therefore, early molecular diagnosis is critical for EVA patients to ensure that the most effective treatment strategies are employed. This study explored a new genetic diagnosis method for EVA and applied it to clinic diagnoses of EVA patients. Using next-generation sequencing technology, we set up a multiple polymerase chain reaction enrichment system for target regions of EVA pathogenic genes (SLC26A4, FOXI1, and KCNJ10). Forty-six EVA samples were sequenced by this system. Variants were detected in 87.0% (40/46) of cases, including three novel variants (SLC26A4 c.923_929del, c.1002-8C>G, and FOXI1 c.519C>A). Biallelic potential pathogenic variants were detected in 27/46 patient samples, leading to a purported diagnostic rate of 59%. All results were verified by Sanger sequencing. Our target region capture system was validated to amplify and measure SLC26A4, FOXI1, and KCNJ10 in one reaction system. The result supplemented the mutation spectrum of EVA. Thus, this strategy is an economic, rapid, accurate, and reliable method with many useful applications in the clinical diagnosis of EVA patients.

Physiological response and sulfur metabolism of the V. dahliae-infected tomato plants in tomato/potato onion companion cropping.

Companion cropping with potato onions (Allium cepa var. agrogatum Don.) can enhance the disease resistance of tomato plants (Solanum lycopersicum) to Verticillium dahliae infection by increasing the expressions of genes related to disease resistance. However, it is not clear how tomato plants physiologically respond to V. dahliae infection and what roles sulfur plays in the disease-resistance. Pot experiments were performed to examine changes in the physiology and sulfur metabolism of tomato roots infected by V. dahliae under the companion cropping (tomato/potato onion). The results showed that the companion cropping increased the content of total phenol, lignin and glutathione and increased the activities of peroxidase, polyphenol oxidase and phenylalanine ammonia lyase in the roots of tomato plants. RNA-seq analysis showed that the expressions of genes involved in sulfur uptake and assimilation, and the formation of sulfur-containing defense compounds (SDCs) were up-regulated in the V. dahlia-infected tomatoes in the companion cropping. In addition, the interactions among tomato, potato onion and V. dahliae induced the expression of the high- affinity sulfate transporter gene in the tomato roots. These results suggest that sulfur may play important roles in tomato disease resistance against V. dahliae.

Nuclear Localised MORE SULPHUR ACCUMULATION1 Epigenetically Regulates Sulphur Homeostasis in Arabidopsis thaliana.

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S-adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over-accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation.

AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis.

Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl(-)-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing.

In mammals, the membrane-based protein Prestin confers unique electromotile properties to cochlear outer hair cells, which contribute to the cochlear amplifier. Like mammals, the ears of insects, such as those of Drosophila melanogaster, mechanically amplify sound stimuli and have also been reported to express Prestin homologs. To determine whether the D. melanogaster Prestin homolog (dpres) is required for auditory amplification, we generated and analyzed dpres mutant flies. We found that dpres is robustly expressed in the fly's antennal ear. However, dpres mutant flies show normal auditory nerve responses, and intact non-linear amplification. Thus we conclude that, in D. melanogaster, auditory amplification is independent of Prestin. This finding resonates with prior phylogenetic analyses, which suggest that the derived motor function of mammalian Prestin replaced, or amended, an ancestral transport function. Indeed, we show that dpres encodes a functional anion transporter. Interestingly, the acquired new motor function in the phylogenetic lineage leading to birds and mammals coincides with loss of the mechanotransducer channel NompC (=TRPN1), which has been shown to be required for auditory amplification in flies. The advent of Prestin (or loss of NompC, respectively) may thus mark an evolutionary transition from a transducer-based to a Prestin-based mechanism of auditory amplification.

Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation.

Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic L-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions.

Identification of high-affinity copper transporters in Aspergillus fumigatus.

We investigated the copper metabolism of Aspergillus fumigatus, which has not been characterized well. We cloned the putative copper transporters ctrA2 and ctrC from A. fumigatus and investigated the functions of these transporters in copper metabolism. Four putative copper transporters were identified in the A. fumigatus genome; ctrA2 and ctrC complemented CTR1 functionally and localized to the plasma membrane in Saccharomyces cerevisiae. ctrA2 and ctrC single-deletion mutants and a double-deletion mutant of ctrA2 and ctrC were constructed in A. fumigatus. The ctrA2 and ctrC double-deletion mutant exhibited a growth defect on Aspergillus minimal medium (AMM) supplemented with bathocuproine disulfonic acid (BCS) and was sensitive to H2O2. Furthermore, the deletion of ctrA2 and ctrC reduced superoxide dismutase (SOD) activity, laccase activity, and intracellular copper contents. The activities of the ctrA2 and ctrC genes were up-regulated by BCS treatment. In addition, the deletion of ctrA2 up-regulated ctrC and vice versa. ctrA2 and ctrC were localized to the A. fumigatus plasma membrane. Although ctrA2 and ctrC failed to affect the mouse survival rate, these genes affected conidial killing activity. Taken together, these results indicate that ctrA2 and ctrC may function as membrane transporters and that the involvement of these genes in pathogenicity merits further investigation.