Low-affinity Transport Research Articles

Structure and sucrose binding mechanism of the plant SUC1 sucrose transporter

Sucrose import from photosynthetic tissues into the phloem is mediated by transporters from the low-affinity sucrose transporter family (SUC/SUT family). Furthermore, sucrose redistribution to other tissues is driven by phloem sap movement, the product of high turgor pressure created by this import activity. Additionally, sink organs such as fruits, cereals and seeds that accumulate high concentrations of sugar also depend on this active transport of sucrose. Here we present the structure of the sucrose–proton symporter, Arabidopsis thaliana SUC1, in an outward open conformation at 2.7 Å resolution, together with molecular dynamics simulations and biochemical characterization. We identify the key acidic residue required for proton-driven sucrose uptake and describe how protonation and sucrose binding are strongly coupled. Sucrose binding is a two-step process, with initial recognition mediated by the glucosyl moiety binding directly to the key acidic residue in a stringent pH-dependent manner. Our results explain how low-affinity sucrose transport is achieved in plants, and pinpoint a range of SUC binders that help define selectivity. Our data demonstrate a new mode for proton-driven symport with links to cation-driven symport and provide a broad model for general low-affinity transport in highly enriched substrate environments.

Analysis of the Biological Activity and Whole Genome Sequencing of Bacillus cereus CDHWZ7 Isolated from the Rhizosphere of Lycium ruthenicum on the Tibetan Plateau

This study examined the biological activity and genome of Bacillus cereus CDHWZ7 isolated from the root of Lycium ruthenicum in the Dachaidan saline area, Haixi Prefecture, Qinghai Province, China. The results revealed that B. cereus CDHWZ7 exhibited strong inhibition activity against the pathogenic fungi Fusarium graminearum, F. acuminatum, and F. oxysporum. CDHWZ7 also demonstrated cellulose-degrading activity, nitrogen-fixing activity, and the ability to secrete indole-3-acetic acid (IAA) at 55.00 mg∙L−1. The strain CDHWZ7 can grow at a salt concentration of 3–11%, a pH range of 5–11, and a temperature of 4 °C–18 °C, and shows good salt tolerance, acid and alkaline tolerance, and low-temperature fitness. The genome of strain CDHWZ7 was sequenced using Illumina HiSeq + PacBio, revealing a circular structure of 5,648,783 bp in length, containing two intact plasmids with an average GC content of 35.2%, and a total number of 5672 encoded genes. It contained 106 tRNA genes, 42 rRNA genes, and 134 sRNA genes. A total of 137 genes were annotated as carbohydrases, with a total base length of 3,968,396,297 bp. The numbers of coding sequences assigned to the Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of Proteins, and Gene Ontology Databases were 4038, 4133, and 2160, respectively. Further analysis of the genome identified genes encoding chitinase activity, cellulases, secondary metabolites, phytohormone production, volatile compounds, nitrogen and phosphate metabolism, and resistance responses to biotic stresses (glycine betaine transporter protein, catalase, superoxide dismutase, low-affinity potassium transporter protein, cold-shock protein, heat-shock protein), as well as genes related to proliferation, stress response, and resistance to pathogenic fungi. Therefore, this study determined that strain CDHWZ7 has several excellent biological traits, such as antagonism to pathogenic fungi, nitrogen-fixation ability, cellulose-degradation ability, and IAA-production ability. The genome sequence of strain CDHWZ7 and several biodefense functional genes were also analyzed, revealing the potential use of strain CDHWZ7 in the development of biological agents.

OsNPF3.1, a nitrate, abscisic acid and gibberellin transporter gene, is essential for rice tillering and nitrogen utilization efficiency

Low-affinity nitrate transporter genes have been identified in subfamilies 4–8 of the rice nitrate transporter 1 (NRT1)/peptide transporter family (NPF), but the OsNPF3 subfamily responsible for nitrate and phytohormone transport and rice growth and development remains unknown. In this study, we described OsNPF3.1 as an essential nitrate and phytohormone transporter gene for rice tillering and nitrogen utilization efficiency (NUtE). OsNPF3.1 possesses four major haplotypes of its promoter sequence in 517 cultivars, and its expression is positively associated with tiller number. Its expression was higher in the basal part, culm, and leaf blade than in other parts of the plant, and was strongly induced by nitrate, abscisic acid (ABA) and gibberellin 3 (GA3) in the root and shoot of rice. Electrophysiological experiments demonstrated that OsNPF3.1 is a pH-dependent low-affinity nitrate transporter, with rice protoplast uptake assays showing it to be an ABA and GA3 transporter. OsNPF3.1 overexpression significantly promoted ABA accumulation in the roots and GA accumulation in the basal part of the plant which inhibited axillary bud outgrowth and rice tillering, especially at high nitrate concentrations. The NUtE of OsNPF3.1-overexpressing plants was enhanced under low and medium nitrate concentrations, whereas the NUtE of OsNPF3.1 clustered regularly interspaced short palindromic repeats (CRISPR) plants was increased under high nitrate concentrations. The results indicate that OsNPF3.1 transports nitrate and phytohormones in different rice tissues under different nitrate concentrations. The altered OsNPF3.1 expression improves NUtE in the OsNPF3.1-overexpressing and CRISPR lines at low and high nitrate concentrations, respectively.

Convergently selected NPF2.12 coordinates root growth and nitrogen use efficiency in wheat and barley.

Understanding the genetic and molecular function of nitrate sensing and acquisition across crop species will accelerate breeding of cultivars with improved nitrogen use efficiency (NUE). Here, we performed a genome-wide scan using wheat and barley accessions characterized under low and high N inputs that uncovered the NPF2.12 gene, encoding a homolog of the Arabidopsis nitrate transceptor NRT1.6 and other low-affinity nitrate transporters that belong to the MAJOR FACILITATOR SUPERFAMILY. Next, it is shown that variations in the NPF2.12 promoter correlated with altered NPF2.12 transcript levels where decreased gene expression was measured under low nitrate availability. Multiple field trials revealed a significantly enhanced N content in leaves and grains and NUE in the presence of the elite allele TaNPF2.12TT grown under low N conditions. Furthermore, the nitrate reductase encoding gene NIA1 was up-regulated in npf2.12 mutant upon low nitrate concentrations, thereby resulting in elevated levels of nitric oxide (NO) production. This increase in NO correlated with the higher root growth, nitrate uptake, and N translocation observed in the mutant when compared to wild-type. The presented data indicate that the elite haplotype alleles of NPF2.12 are convergently selected in wheat and barley that by inactivation indirectly contribute to root growth and NUE by activating NO signaling under low nitrate conditions.

A study on L-threonine and L-serine uptake in Escherichia coli K-12.

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

Transcriptome and Low-Affinity Sodium Transport Analysis Reveals Salt Tolerance Variations between Two Poplar Trees.

Salinity stress severely hampers plant growth and productivity. How to improve plants' salt tolerance is an urgent issue. However, the molecular basis of plant resistance to salinity still remains unclear. In this study, we used two poplar species with different salt sensitivities to conduct RNA-sequencing and physiological and pharmacological analyses; the aim is to study the transcriptional profiles and ionic transport characteristics in the roots of the two Populus subjected to salt stress under hydroponic culture conditions. Our results show that numerous genes related to energy metabolism were highly expressed in Populus alba relative to Populus russkii, which activates vigorous metabolic processes and energy reserves for initiating a set of defense responses when suffering from salinity stress. Moreover, we found the capacity of Na+ transportation by the P. alba high-affinity K+ transporter1;2 (HKT1;2) was superior to that of P. russkii under salt stress, which enables P. alba to efficiently recycle xylem-loaded Na+ and to maintain shoot K+/Na+ homeostasis. Furthermore, the genes involved in the synthesis of ethylene and abscisic acid were up-regulated in P. alba but downregulated in P. russkii under salt stress. In P. alba, the gibberellin inactivation and auxin signaling genes with steady high transcriptions, several antioxidant enzymes activities (such as peroxidase [POD], ascorbate peroxidase [APX], and glutathione reductase [GR]), and glycine-betaine content were significantly increased under salt stress. These factors altogether confer P. alba a higher resistance to salinity, achieving a more efficient coordination between growth modulation and defense response. Our research provides significant evidence to improve the salt tolerance of crops or woody plants.

Integration of Transcriptome and Metabolome Analyses Reveals the Mechanistic Basis for Cadmium Accumulation in Maize.

Cadmium (Cd) pollution in soil has become a major environmental issue worldwide. However, the underlying molecular mechanism of low grain-Cd accumulation (GCA) in maize is still largely unknown. Herein, we report the mechanistic basis for low GCA in maize by a multiomics approach. The low GCA genotype L63 showed normal vacuolar formation and a lower capacity of xylem loading of Cd than the high-accumulator L42 under Cd stress. Transcriptomic sequencing identified 84 low-GCA-associated genes which are mainly involved in the S-adenosylmethionine (SAM) cycle, metal transport, and vacuolar sequestration. A metabolome analysis revealed that L63 plants had a more active SAM cycle and a greater capacity for terpenoid synthesis and phenylalanine metabolism than L42. Combining the analysis of transcriptome and metabolome characterized several genes as key genes involved in the determination of Cd accumulation. Our study identifies a mechanistic basis for low Cd accumulation in maize grains and provides candidate genes for genetic improvement of crops.

Lack of Low-affinity Phosphate Transporter Pho91 Alters Lipid Metabolism in Yeast Saccharomyces cerevisiae

Objective: The current study tries to elucidate the impact of low-affinity Pi transporter Pho91 (using pho91Δ cells) on lipid metabolism and cellular organelles morphology (mitochondria and vacuole) in yeast Saccharomyces cerevisiae. Methods: The lipid profile was performed using thin layer chromatography (TLC), and the membrane defect was determined using DiOC6 staining, lipid droplets (LDs) were observed by Nile red staining, mitochondrial morphology was observed using aconitase 1 GFP, vacuolar morphology was studied by FM4-64 staining and were performed with the aid of laser scanning fluorescent microscope. Findings: Pho91 is a low-affinity phosphate (Pi) transporter in the vacuoles that functions under Pi-rich conditions, but its role in lipid metabolism is largely unknown. In this study, we used defined synthetic complete media (SC). The deletion of Pho91 depicted a moderate growth defect but increased the major phospholipids PC, PE, and PI. Alterations in the membrane phospholipids resulted in defective membrane morphology. In pho91 mutant (pho91Δ), the neutral lipids TAG and SE were increased and stored as LDs. The LD numbers were increased in pho91Δ cells than in WT cells. Altered phospholipids also defective mitochondrial morphology and enlarged vacuoles in pho91 deletion. Novelty: In the absence of low-affinity Pi transporter pho91, it increases phospholipid, neutral lipid levels, LD numbers, and impacted mitochondria along with vacuolar structures. Keywords: Phosphate transporter; Low affinity; Phospholipids; Neutral lipids; Lipid droplets

The cassava (Manihot-esculenta Crantz)'s nitrate transporter NPF4.5, expressed in seedling roots, involved in nitrate flux and osmotic stress

AtNPF4.5/AIT2, which was predicted to be a low-affinity transporter capable for nitrate uptake, was screened by ABA receptor complex in Arabidopsis ten years ago. However, the molecular and biochemical characterizations of AtNPF4.5 in plants remained largely unclear. In this study, the function of a plasma-membrane-localized and root-specifically-expressed gene MeNPF4.5 (Manihot-esculenta NITRATE TRANSPORTER 1 PTR FAMILY4.5), an ortholog of the Arabidopsis thaliana NPF4.5, was investigated in cassava roots as a nitrate efflux transporter on low nitrate medium and an influx transporter following exposure to high concentration of external nitrates. Moreover, RNA interference (RNAi) of MeNPF4.5 reduced the nitrate efflux capacity but the overexpressing cassava seedlings increased the ability of efflux from the elongation to the mature zone of root under low nitrate treatments. Besides, MeNPF4.5-RNAi expression reduced the nitrate influx capacity but enhanced nitrate absorption in parts of overexpressing plants from the meristem, elongation to mature zone of roots under high nitrate conditions. Furthermore, MeNPF4.5-RNAi seedlings survived owing to roots that could grow normally, but the MeNPF4.5-over-expressors showed adverse growth under 7% PEG6000 stress, suggesting that MeNPF4.5 negatively regulated the osmotic stress and was involved in nitrate flux through cassava seedlings.

Vacuolar nitrate efflux requires multiple functional redundant nitrate transporter in Arabidopsis thaliana.

Nitrate in plants is preferentially stored in vacuoles; however, how vacuolar nitrate is reallocated and to which biological process(es) it might contribute remain largely elusive. In this study, we functionally characterized three nitrate transporters NPF5.10, NPF5.14, and NPF8.5 that are tonoplast-localized. Ectopic expression in Xenopus laevis oocytes revealed that they mediate low-affinity nitrate transport. Histochemical analysis showed that these transporters were expressed preferentially in pericycle and xylem parenchyma cells. NPF5.10, NPF5.14, and NPF8.5 overexpression significantly decreased vacuolar nitrate contents and nitrate accumulation in Arabidopsis shoots. Further analysis showed that the sextuple mutant (npf5.10 npf5.14 npf8.5 npf5.11 npf5.12 npf5.16) had a higher 15NO3-uptake rate than the wild-type Col-0, but no significant difference was observed for nitrate accumulation between them. The septuple mutant (npf5.11 npf5.12 npf5.16 npf5.10 npf5.14 npf8.5 clca) generated by using CRISPR/Cas9 showed essentially decreased nitrate reallocation compared to wild type when exposed to nitrate starvation, though no further decrease was observed when compared to clca. Notably, NPF5.10, NPF5.14, and NPF8.5 as well as NPF5.11, NPF5.12, and NPF5.16 were consistently induced by mannitol, and more nitrate was detected in the sextuple mutant than in the wild type after mannitol treatment. These observations suggest that vacuolar nitrate efflux is regulated by several functional redundant nitrate transporters, and the reallocation might contribute to osmotic stress response other than mineral nutrition.

A controversial issue: Can mitochondria modulate cytosolic calcium and contraction of skeletal muscle fibers?

Mitochondria are characterized by a high capacity to accumulate calcium thanks to the electrochemical gradient created by the extrusion of protons in the respiratory chain. Thereby calcium can enter crossing the inner mitochondrial membrane via MCU complex, a high-capacity, low-affinity transport mechanism. Calcium uptake serves numerous purposes, among them the regulation of three dehydrogenases of the citric cycle, apoptosis via permeability transition, and, in some cell types, modulation of cytosolic calcium transients. This Review is focused on mitochondrial calcium uptake in skeletal muscle fibers and aims to reanalyze its functional impact. In particular, we ask whether mitochondrial calcium uptake is relevant for the control of cytosolic calcium transients and therefore of contractile performance. Recent data suggest that this may be the case, at least in particular conditions, as modified expression of MCU complex subunits or of proteins involved in mitochondrial dynamics and ablation of the main cytosolic calcium buffer, parvalbumin.

Genome-wide identification and in silico analysis of NPF, NRT2, CLC and SLAC1/SLAH nitrate transporters in hexaploid wheat (Triticum aestivum)

Nitrogen transport is one of the most important processes in plants mediated by specialized transmembrane proteins. Plants have two main systems for nitrogen uptake from soil and its transport within the system—a low-affinity transport system and a high-affinity transport system. Nitrate transporters are of special interest in cereal crops because large amount of money is spent on N fertilizers every year to enhance the crop productivity. Till date four gene families of nitrate transporter proteins; NPF (nitrate transporter 1/peptide transporter family), NRT2 (nitrate transporter 2 family), the CLC (chloride channel family), and the SLAC/SLAH (slow anion channel-associated homologues) have been reported in plants. In our study, in silico mining of nitrate transporter genes along with their detailed structure, phylogenetic and expression analysis was carried out. A total of 412 nitrate transporter genes were identified in hexaploid wheat genome using HMMER based homology searches in IWGSC Refseq v2.0. Out of those twenty genes were root specific, 11 leaf/shoot specific and 17 genes were grain/spike specific. The identification of nitrate transporter genes in the close proximity to the previously identified 67 marker-traits associations associated with the nitrogen use efficiency related traits in nested synthetic hexaploid wheat introgression library indicated the robustness of the reported transporter genes. The detailed crosstalk between the genome and proteome and the validation of identified putative candidate genes through expression and gene editing studies may lay down the foundation to improve nitrogen use efficiency of cereal crops.

Crosstalk Between Iron and Sulfur Homeostasis Networks in Arabidopsis.

The widespread deficiency of iron (Fe) and sulfur (S) is becoming a global concern. The underlying mechanisms regulating Fe and S sensing and signaling have not been well understood. We investigated the crosstalk between Fe and S using mutants impaired in Fe homeostasis, sulfate assimilation, and glutathione (GSH) biosynthesis. We showed that chlorosis symptoms induced by Fe deficiency were not directly related to the endogenous GSH levels. We found dynamic crosstalk between Fe and S networks and more interestingly observed that the upregulated expression of IRT1 and FRO2 under S deficiency in Col-0 was missing in the cad2-1 mutant background, which suggests that under S deficiency, the expression of IRT1 and FRO2 was directly or indirectly dependent on GSH. Interestingly, the bottleneck in sulfite reduction led to a constitutively higher IRT1 expression in the sir1-1 mutant. While the high-affinity sulfate transporter (Sultr1;2) was upregulated under Fe deficiency in the roots, the low-affinity sulfate transporters (Sultr2;1, and Sultr2;2) were down-regulated in the shoots of Col-0 seedlings. Moreover, the expression analysis of some of the key players in the Fe–S cluster assembly revealed that the expression of the so-called Fe donor in mitochondria (AtFH) and S mobilizer of group II cysteine desulfurase in plastids (AtNFS2) were upregulated under Fe deficiency in Col-0. Our qPCR data and ChIP-qPCR experiments suggested that the expression of AtFH is likely under the transcriptional regulation of the central transcription factor FIT.

Anaerobic ATP synthesis pathways and inorganic phosphate transport and their possible roles in encystment in Acanthamoeba castellanii

Acanthamoeba castellanii is the etiological agent of amoebic keratitis and is present in the environment in trophozoite or cyst forms. Both forms can infect the vertebrate host and colonize different tissues. The high resistance of cysts to standard drugs used in clinics contributes to the lack of effective treatments. Therefore, in this context, studies have emerged to understand cyst physiology and metabolism. Phosphate transporters are proteins responsible for the uptake of extracellular inorganic phosphate and transport to the cytosol. This work aims to verify the relationship between Pi transport and energetic metabolism in cysts of A. castellanii. The phosphate uptake ratio was higher in cysts compared with trophozoites. Recently, three sequences related to phosphate transporters have been identified in the A. castellanii genome (AcPHS1, AcPHS2, and AcPHS3); the messenger RNA expression levels of which differ depending on the amoeba life form. Pi uptake in cysts displayed peak activity at alkaline pH, whereas Pi transport in trophozoites was not affected in the same pH ranges. Cysts harbor a low-affinity Pi transport system (K0,5 and Vmax values of 1.76 ± 0.26 mM and 104.6 ± 6.3 nmol Pi × h-1 × 106 cells) compared to the trophozoite phosphate transport system. Pi transport seems important for anaerobic adenosine triphosphate synthesis in cysts, which initially occurs through the glycolytic pathway and subsequently through the pyruvate ferredoxin oxidoreductase pathway. Altogether, these results suggest that contrary to that previously postulated, cysts are active metabolic forms, and, as noted in trophozoites, phosphate uptake is important for energetic metabolism.

Effects of Different Inorganic Nitrogen Sources of Iris pseudacorus and Iris japonica on Energy Distribution, Nitrogen, and Phosphorus Removal

High- and low-affinity transport systems are the main pathways for the transportation of NO3– and NH4+ across intracellular membranes. NO3− and NH4+ are assimilated through different metabolic pathways in plants. Fifteen ATP molecules are hydrolyzed in the metabolic process of NO3–; however, only five ATP molecules are hydrolyzed in that of NH4+. In this research, seedlings of Iris pseudacorus and Iris japonica were used as the experimental materials in the NO3–:NH4+ = 30:0, NO3–:NH4+ = 28:2, NO3–:NH4+ = 27:3, NO3–:NH4+ = 15:15, NO3–:NH4+ = 3:27, and NO3–:NH4+ = 0:30 treatments at the 7.5 mmol·L−1 the total nitrogen content (TN). The intracellular free energy was represented by physiological resistance (R) and physiological impedance (Z) according to the Nernst equation and could conveniently and comprehensively determine the cellular metabolic energy (GB). The maximum absorption rate (Vmax) and Michaelis constant (Km) for NH4+ and NO3– uptake were calculated according to the kinetic equation. The results showed that the cellular metabolic energy (GB) of I. pseudacorus was 1 to 1.5 times lower than that of I. japonica at each treatment on the 10th day. The GB values of I. pseudacorus and I. japonica seedlings increased with increasing NH4+ concentration. However, there was a turning point at the NO3–:NH4+ = 15:15 treatment for the cellular metabolic energy of I. pseudacorus and I. japonica. Correlation analysis showed that the value of cellular metabolic energy was negatively correlated with the Vmax and Km for NO3– uptake, whereas it was positively correlated with that for NH4+ uptake. These results demonstrate that the NO3–:NH4+ = 27:3 treatment level was the most suitable for I. pseudacorus and I. japonica. This indicates that the greater cellular metabolic energy is the most suitable for plant growth when the concentration of ammonium or nitrate had no significant difference at treatment. These results provide a simple and rapid solution for removal of nitrogen by determination of cellular metabolic energy.

Analysing and meta-analysing time-series data of microbial growth and gene expression from plate readers.

Responding to change is a fundamental property of life, making time-series data invaluable in biology. For microbes, plate readers are a popular, convenient means to measure growth and also gene expression using fluorescent reporters. Nevertheless, the difficulties of analysing the resulting data can be a bottleneck, particularly when combining measurements from different wells and plates. Here we present omniplate, a Python module that corrects and normalises plate-reader data, estimates growth rates and fluorescence per cell as functions of time, calculates errors, exports in different formats, and enables meta-analysis of multiple plates. The software corrects for autofluorescence, the optical density’s non-linear dependence on the number of cells, and the effects of the media. We use omniplate to measure the Monod relationship for the growth of budding yeast in raffinose, showing that raffinose is a convenient carbon source for controlling growth rates. Using fluorescent tagging, we study yeast’s glucose transport. Our results are consistent with the regulation of the hexose transporter (HXT) genes being approximately bipartite: the medium and high affinity transporters are predominately regulated by both the high affinity glucose sensor Snf3 and the kinase complex SNF1 via the repressors Mth1, Mig1, and Mig2; the low affinity transporters are predominately regulated by the low affinity sensor Rgt2 via the co-repressor Std1. We thus demonstrate that omniplate is a powerful tool for exploiting the advantages offered by time-series data in revealing biological regulation.

Comparative Genomics of Members of the Genus Defluviicoccus With Insights Into Their Ecophysiological Importance.

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca. Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published (Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca. Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanusT, the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus.

Mammalian monocarboxylate transporter 7 (MCT7/Slc16a6) is a novel facilitative taurine transporter

Monocarboxylate transporter 7 (MCT7) is an orphan transporter expressed in the liver, brain, and in several types of cancer cells. It has also been reported to be a survival factor in melanoma and breast cancers. However, this survival mechanism is not yet fully understood due to MCT7’s unidentified substrate(s). Therefore, here we sought to identify MCT7 substrate(s) and characterize the transport mechanisms by analyzing amino acid transport in HEK293T cells and polarized Caco-2 cells. Analysis of amino acids revealed significant rapid reduction in taurine from cells transfected with enhanced green fluorescent protein-tagged MCT7. We found that taurine uptake and efflux by MCT7 was pH-independent and that the uptake was not saturated in the presence of taurine excess of 200 mM. Furthermore, we found that monocarboxylates and acidic amino acids inhibited MCT7-mediated taurine uptake. These results imply that MCT7 may be a low-affinity facilitative taurine transporter. We also found that MCT7 was localized at the basolateral membrane in polarized Caco-2 cells and that the induction of MCT7 expression in polarized Caco-2 cells enhanced taurine permeation. Finally, we demonstrated that interactions of MCT7 with ancillary proteins basigin/CD147 and embigin/GP70 enhanced MCT7-mediated taurine transport. In summary, these findings reveal that taurine is a novel substrate of MCT7 and that MCT7-mediated taurine transport might contribute to the efflux of taurine from cells.

The nitrate transporter OsNPF7.9 mediates nitrate allocation and the divergent nitrate use efficiency between indica and japonica rice.

Nitrate allocation in Arabidopsis (Arabidopsis thaliana) represents an important mechanism for mediating plant environmental adaptation. However, whether this mechanism occurs or has any physiological/agronomic importance in the ammoniphilic plant rice (Oriza sativa L.) remains unknown. Here, we address this question through functional characterization of the Nitrate transporter 1/Peptide transporter Family (NPF) transporter gene OsNPF7.9. Ectopic expression of OsNPF7.9 in Xenopus oocytes revealed that the gene encodes a low-affinity nitrate transporter. Histochemical and in-situ hybridization assays showed that OsNPF7.9 expresses preferentially in xylem parenchyma cells of vasculature tissues. Transient expression assays indicated that OsNPF7.9 localizes to the plasma membrane. Nitrate allocation from roots to shoots was essentially decreased in osnpf7.9 mutants. Biomass, grain yield, and nitrogen use efficiency (NUE) decreased in the mutant dependent on nitrate availability. Further analysis demonstrated that nitrate allocation mediated by OsNPF7.9 is essential for balancing rice growth and stress tolerance. Moreover, our research identified an indica-japonica divergent single-nucleotide polymorphism occurring in the coding region of OsNPF7.9, which correlates with enhanced nitrate allocation to shoots of indica rice, revealing that divergent nitrate allocation might represent an important component contributing to the divergent NUE between indica and japonica subspecies and was likely selected as a favorable trait during rice breeding.

Identification and analysis of iron transporters from the fission yeast Schizosaccharomyces pombe.

Iron is an essential trace metal ion required for all living organisms, and is taken up by iron transporters. Here, we identified and characterized three-candidate high-affinity (Fio1, Frp1 and Frp2) and two-candidate low-affinity iron transporters (Fet4 and Pdt1) from the fission yeast Schizosaccharomyces pombe. Protein sequence analyses revealed that Fio1 is a multicopper oxidase that contains three cupredoxin domains with eleven candidate iron-binding ligands, whereas Frp1 harbors a ferric reductase domain with three-candidate heme-binding ligands. Protein sequence analyses also revealed that Fet4 and Pdt1 are integral membrane proteins with 10 and 11 transmembrane regions, respectively. Deletion of fio1 and, to a lesser extent, frp1 impaired growth under iron-depleted conditions, whereas deletion of frp1 and, to a lesser extent, frp2 inhibited growth under iron-replete conditions. Deletion of fet4 and pdt1 did not affect the growth of cells under iron-depleted and iron-replete conditions. Deletion of fio1 or frp1 also increased the sensitivity of cells to other transition metals. The copper sensitivity of Δfio1 cells could be rescued by iron, suggesting that the addition of iron mightdecrease the uptake of potentially toxic copper in Δfio1 cells. The copper sensitivity of Δfio1 cells could also be rescued by deletion of frp1, suggesting that Fio1 and Frp1 may function together in iron and copper uptakes in S. pombe. Our results revealed that iron and copper uptake systems may be partially overlapped in S. pombe.