Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

Interactions of cytosolic tails in the Jen1 carboxylate transporter are critical for trafficking and transport activity.

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism, growth and response to stress or drugs. In Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that provides a model to dissect the molecular details underlying cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence revealing novel roles of the cytosolic N- and C-termini of Jen1 in its biogenesis, PM stability and transport activity, using functional analyses of Jen1 truncations and chimeric constructs with UapA, an endocytosis-insensitive transporter of Aspergillus nidulans. Our results show that both N- and C-termini are critical for Jen1 trafficking to the PM, transport activity and endocytosis. Importantly, we provide evidence that Jen1 N- and C-termini undergo transport-dependent dynamic intramolecular interactions, which affect the transport activity and turnover of Jen1. Our results support an emerging concept where the cytoplasmic termini of PM transporters control transporter cell surface stability and function through flexible intramolecular interactions with each other. These findings might be extended to other MFS members to understand conserved and evolving mechanisms underlying transporter structure-function relationships. This article has an associated First Person interview with the first authors of the paper.

Correlating sugar transporter expression and activities to identify transporters for an orphan sugar substrate.

Filamentous fungi like Neurospora crassa are able to take up and metabolize important sugars present, for example, in agricultural and human food wastes. However, only a fraction of all putative sugar transporters in filamentous fungi has been characterized to date, and for many sugar substrates, the corresponding transporters are unknown. In N. crassa, only 14 out of the 42 putative major facilitator superfamily (MFS)-type sugar transporters have been characterized so far. To uncover this hidden potential for biotechnology, it is therefore necessary to find new strategies. By correlation of the uptake profile of sugars of interest after different induction conditions with the expression profiles of all 44 genes encoding predicted sugar transporters in N. crassa, together with an exhaustive phylogenetic analysis using sequences of characterized fungal sugar transporters, we aimed to identify transporter candidates for the tested sugars. Following this approach, we found a high correlation of uptake rates and expression strengths for many sugars with dedicated transporters, like galacturonic acid and arabinose, while the correlation is loose for sugars that are transported by several transporters due to functional redundancy. Nevertheless, this combinatorial approach allowed us to elucidate the uptake system for the disaccharide lactose, a by-product of the dairy industry, which consists of the two main cellodextrin transporters CDT-1 and CDT-2 with a minor contribution of the related transporter NCU00809. Moreover, a non-MFS transporter involved in glycerol transport was also identified. Deorphanization of sugar transporters or identification of transporters for orphan sugar substrates by correlation of uptake kinetics with transporter expression and phylogenetic information can thus provide a way to optimize the reuse of food industry by-products and agricultural wastes by filamentous fungi in order to create economic value and reduce their environmental impact. KEY POINTS: • The Neurospora crassa genome contains 30 uncharacterized putative sugar transporter genes. • Correlation of transporter expression and sugar uptake profiles can help to identify transporters for orphan sugar substrates. • CDT-1, CDT-2, and NCU00809 are key players in the transport of the dairy by-product lactose in N. crassa.

Key computational findings reveal proton transfer as driving the functional cycle in the phosphate transporter PiPT.

Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.

AadT, a new weapon in Acinetobacter's fight against antibiotics.

Genes encoding a novel multidrug efflux pump, AadT, from the Drug:H+ antiporter 2 family, were discovered in Acinetobacter multidrug resistance plasmids. Here, we profiled the antimicrobial resistance potential, and examined the distribution of these genes. aadT homologs were found in many Acinetobacter and other Gram-negative species and were typically adjacent to novel variants of adeAB(C), which encodes a major tripartite efflux pump in Acinetobacter. The AadT pump decreased bacterial susceptibility to at least eight diverse antimicrobials, including antibiotics (erythromycin and tetracycline), biocides (chlorhexidine), and dyes (ethidium bromide and DAPI) and was able to mediate ethidium transport. These results show that AadT is a multidrug efflux pump in the Acinetobacter resistance arsenal and may cooperate with variants of AdeAB(C).

Identification of a specific exporter that enables high production of aconitic acid in Aspergillus pseudoterreus.

Aconitic acid is an unsaturated tricarboxylic acid that is attractive for its potential use in manufacturing biodegradable and biocompatible polymers, plasticizers, and surfactants. Previously Aspergillus pseudoterreus was engineered as a platform to produce aconitic acid by deleting the cadA (cis-aconitic acid decarboxylase) gene in the itaconic acid biosynthetic pathway. In this study, the aconitic acid transporter gene (aexA) was identified using comparative global discovery proteomics analysis between the wild-type and cadA deletion strains. The protein AexA belongs to the Major Facilitator Superfamily (MFS). Deletion of aexA almost abolished aconitic acid secretion, while its overexpression led to a significant increase in aconitic acid production. Transportation of aconitic acid across the plasma membrane is a key limiting step in its production. In vitro, proteoliposome transport assay further validated AexA's function and substrate specificity. This research provides new approaches to efficiently pinpoint and characterize exporters of fungal organic acids and accelerate metabolic engineering to improve secretion capability and lower the cost of bioproduction.

Hypertension depresses but exercise training restores both Mfsd2a expression and blood-brain barrier function within PVN capillaries.

Hypertension augments while exercise training corrects the increased vesicle trafficking (transcytosis) across the blood-brain barrier (BBB) within preautonomic areas and the autonomic imbalance. There is no information on a possible mechanism(s) conditioning these effects. Knowing that Mfsd2a is the major transporter of docosahexaenoic acid (DHA) and that Mfsd2a knockout mice exhibited leaky BBB, we sought to identify its possible involvement in hypertension- and exercise-induced transcytosis across the BBB. Spontaneously hypertensive rats (SHR) and Wistar rats were submitted to treadmill training (T) or kept sedentary (S) for 4 wk. Resting hemodynamic/autonomic parameters were recorded in conscious chronically cannulated rats. BBB permeability within the hypothalamic paraventricular nucleus (PVN) was evaluated in anesthetized rats. Brains were harvested for Mfsd2a and caveolin-1 (an essential protein for vesicle formation) expression. SHR-S versus Wistar-S exhibited elevated arterial pressure (AP) and heart rate (HR), increased vasomotor sympathetic activity, reduced cardiac parasympathetic activity, greater pressure variability, reduced HR variability, and depressed baroreflex control. SHR-S also showed increased BBB permeability, reduced Mfsd2a, and increased caveolin-1 expression. SHR-T versus SHR-S exhibited increased Mfsd2a density, reduced caveolin-1 protein expression, and normalized PVN BBB permeability, which were accompanied by resting bradycardia, partial AP drop, reduced sympathetic and normalized cardiac parasympathetic activity, increased HR variability, and reduced pressure variability. No changes were observed in Wistar-T versus Wistar-S. Training is an efficient tool to rescue Mfsd2a expression, which by transporting DHA into the endothelial cell reduces caveolin-1 availability and vesicles' formation. Exercise-induced Mfsd2a normalization is an important mechanism to correct both BBB function and autonomic control in hypertensive subjects.

The evolving biology of the proton-coupled folate transporter: New insights into regulation, structure, and mechanism.

The human proton-coupled folate transporter (PCFT; SLC46A1) or hPCFT was identified in 2006 as the principal folate transporter involved in the intestinal absorption of dietary folates. A rare autosomal recessive hereditary folate malabsorption syndrome is attributable to human SLC46A1 variants. The recognition that hPCFT was highly expressed in many tumors stimulated substantial interest in its potential for cytotoxic drug targeting, taking advantage of its high-level transport activity under acidic pH conditions that characterize many tumors and its modest expression in most normal tissues. To better understand the basis for variations in hPCFT levels between tissues including human tumors, studies have examined the transcriptional regulation of hPCFT including the roles of CpG hypermethylation and critical transcription factors and cis elements. Additional focus involved identifying key structural and functional determinants of hPCFT transport that, combined with homology models based on structural homologies to the bacterial transporters GlpT and LacY, have enabled new structural and mechanistic insights. Recently, cryo-electron microscopy structures of chicken PCFT in a substrate-free state and in complex with the antifolate pemetrexed were reported, providing further structural insights into determinants of (anti)folate recognition and the mechanism of pH-regulated (anti)folate transport by PCFT. Like many major facilitator proteins, hPCFT exists as a homo-oligomer, and evidence suggests that homo-oligomerization of hPCFT monomeric proteins may be important for its intracellular trafficking and/or transport function. Better understanding of the structure, function and regulation of hPCFT should facilitate the rational development of new therapeutic strategies for conditions associated with folate deficiency, as well as cancer.

Quorum Sensing Regulation of a Major Facilitator Superfamily Transporter Affects Multiple Streptococcal Virulence Factors.

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or group A strep (GAS), one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes, including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit, and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. IMPORTANCE The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple virulence factors and accounts for QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

Molecular insights into the differential efflux mechanism of Rv1634 protein, a multidrug transporter of major facilitator superfamily in Mycobacterium tuberculosis.

Currently, multidrug-resistant tuberculosis (MDR-TB) is a public health crisis and a major health security threat globally. In Mycobacterium tuberculosis (Mtb), major facilitator superfamily (MFS) is the largest group of secondary active transporters. Along with the transport of their natural substrates, MFS proteins were involved in a drug efflux mechanism that ultimately lead to resistance against available anti-TB drugs in Mtb. In the present study, the three-dimensional structure model of an MFS protein, Rv1634, a probable multidrug transporter from Mtb, was generated using homology modeling. The protein structure model was found in inward-open conformation having 14 transmembrane helices. In addition, a central transport channel was deduced across the protein, and a single binding pocket was identified halfway through the central cavity by structural alignment with the homologous protein structures. Further, Rv1634 protein was studied based on the differential structural behavior of apo and ligand-bound forms. All the protein systems were inserted into a phospholipid bilayer to characterize the conformational dynamics of the protein using molecular dynamics (MD) simulations. Detailed analysis of the MD trajectories showed the diverse substrate specificity of the binding pocket for the antibiotics that caused differential movement in the ciprofloxacin and norfloxacin, to which Mtb strains have now become resistant. The expulsion of the drugs outside the bacterial cell occurs through the alternating-access mechanism of N and C-terminal domains, which is intriguing and essential to the understanding the drug resistance mechanism in pathogenic bacteria.

Identification of a major facilitator superfamily protein that is beneficial to L-lactic acid production by Bacillus coagulans at low pH.

BACKGROUND: Product inhibition is one of the major problems in lactic acid (LA) fermentation. Our previous study revealed that Bacillus coagulans 2-6 was an efficient producer of high-optical-purity L-LA. Its mutant strain B. coagulans Na-2 has better resistance to sodium lactate stress but the resistance mechanism has not been understood. RESULTS: In this study, the whole-genome sequencing of B. coagulans Na-2 was performed and one mutant gene mfs coding for the major facilitator superfamily (MFS) protein was revealed by comparative genome analysis. Ten mutation sites were identified between the wild (MFS-2-6) and mutant (MFS-Na-2) proteins, among which T127A and N154T were predicted locating in the center of the transmembrane transport channel. The MFS-2-6 and MFS-Na-2 were expressed separately in a genetically operable strain, B. coagulans DSM1, using the genes' native promoter. The expression of the two MFS proteins had no effect and a negative effect on L-LA production when the pH was controlled at 6.0 and 7.0 by sodium hydroxide, respectively. However, 4.2 and 4.6-fold of L-LA concentrations were obtained at pH 5.0 by the strains expressing MFS-2-6 and MFS-Na-2 than that by the control strain, respectively. The intracellular pH values of the strains expressing MFS-2-6 and MFS-Na-2 were approximately 0.69 and 0.45 higher than that of the control strain during pH-controlled fermentation at 5.0. Results suggest that the expression of MFS-2-6 and MFS-Na-2 were both conducive to L-LA production at low pH, while the better performance of the latter was probably due to the more appropriate intracellular pH during the whole fermentation process. CONCLUSIONS: The MFS protein identified here can improve the ability of B. coagulans to resist acidic environments and produce more L-LA at low pH. The MFS protein has an application potential in environment-friendly L-LA production.

Opposite motion of the Central Helices of efflux pump KmrA is important for its export efficiency.

Efflux pump of Major Facilitator Superfamily (MFS) is widely distributed in bacteria, while its role in regulating antibiotic resistance of nosocomial pathogen Klebsiella pneumoniae remains unclear. Herein we analyzed the effect of amino acid substitution of MFS efflux pump KmrA on its export efficiency via molecular biology and molecular dynamics (MD). After searching across the 804 sequenced K. pneumoniae isolates, we identified four major variants of KmrA, while one of them KmrA-A was demonstrated an inactive one in MIC and ethidium bromide efflux assays. Subsequently, MD simulations of KmrA and its variants were conducted and the opposite motion of the central helices were observed for the active variants, while it was not found for KmrA-A. To further identify the importance of the opposite motion to the conformational transition, we calculated their differences in volume of binding pocket, salt bridge and hydrophilic interaction with water based on the rocker-switch model. Our results indicated that the opposite motion of KmrA conferred a larger binding pocket and stronger hydrogen bond with water at inward-facing conformation. An unusual substitution S374A of KmrA-A disrupted the normal motion of central helices by enhancing hydrophobic interactions between them, resulting into the altered positions and strengths of salt bridge, which was deduced to affect the conformational transition. Overall our data provided detailed information on the regular of KmrA's moving trajectory, demonstrating the importance of opposite motion of central helices to KmrA's export efficiency.

Molecular Dynamics Investigation of MFS Efflux Pump MdfA Reveals an Intermediate State between Its Inward and Outward Conformations.

Multidrug resistance poses a major challenge to antibiotic therapy. A principal cause of antibiotic resistance is through active export by efflux pumps embedded in the bacterial membrane. Major facilitator superfamily (MFS) efflux pumps constitute a major group of transporters, which are often related to quinolone resistance in clinical settings. Although a rocker-switch model is proposed for description of their conformational transitions, detailed changes in this process remain poorly understood. Here we used MdfA from E. coli as a representative MFS efflux pump to investigate factors that can affect its conformational transition in silico. Molecular dynamics (MD) simulations of MdfA's inward and outward conformations revealed an intermediate state between these two conformations. By comparison of the subtle differences between the intermediate state and the average state, we indicated that conformational transition from outward to inward was initiated by protonation of the periplasmic side. Subsequently, hydrophilic interaction of the periplasmic side with water was promoted and the regional structure of helix 1 was altered to favor this process. As the hydrophobic interaction between MdfA and membrane was also increased, energy was concentrated and stored for the opposite transition. In parallel, salt bridges at the cytoplasmic side were altered to lower probabilities to facilitate the entrance of substrate. In summary, we described the total and local changes during MdfA's conformational transition, providing insights for the development of potential inhibitors.

Isolation and structural characterization of a Zn2+-bound single-domain antibody against NorC, a putative multidrug efflux transporter in bacteria.

Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14TM helix transporter, NorC, from Staphylococcus aureus We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings.

Asp22 drives the protonation state of the Staphylococcus epidermidis glucose/H+ symporter.

The Staphylococcus epidermidis glucose/H+ symporter (GlcPSe) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H+ symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H+/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H+ co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcPSe We found that Asp22 has a pKa of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcPSe A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp22 We expected that the substitution of the residue Ile105 for a serine, located within hydrogen-bonding distance to Asp22, would change the microenvironment, but the pKa of Asp22 corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp22, with the latter now exhibiting a pKa of 6.4. These studies confirm that Asp22 is the proton-binding residue in GlcPSe and show that charged residues in its vicinity affect the pKa of glucose/H+ symport.

Arabidopsis PIC30 encodes a major facilitator superfamily transporter responsible for the uptake of picolinate herbicides.

Picloram is an auxinic herbicide that is widely used for controlling broad leaf weeds. However, its mechanism of transport into plants is poorly understood. In a genetic screen for picloram resistance, we identified three Arabidopsis mutant alleles of PIC30 (PICLORAM RESISTANT30) that are specifically resistant to picolinates, but not to other auxins. PIC30 is a previously uncharacterized gene that encodes a major facilitator superfamily (MFS) transporter. Similar to most members of MFS, PIC30 contains 12 putative transmembrane domains, and PIC30-GFP fusion protein selectively localizes to the plasma membrane. In planta transport assays demonstrate that PIC30 specifically transports picloram, but not indole-3-acetic acid (IAA). Functional analysis of Xenopus laevis oocytes injected with PIC30 cRNA demonstrated PIC30 mediated transport of picloram and several anions, including nitrate and chloride. Consistent with these roles of PIC30, three allelic pic30 mutants are selectively insensitive to picolinate herbicides, while pic30-3 is also defective in chlorate (analogue of nitrate) transport and also shows reduced uptake of 15NO3- . Overexpression of PIC30 fully complements both picloram and chlorate insensitive phenotypes of pic30-3. Despite the continued use of picloram as an herbicide, a transporter for picloram was not known until now. This work provides insight into the mechanisms of plant resistance to picolinate herbicides and also shed light on the possible endogenous function of PIC30 protein.

CgMFS1, a Major Facilitator Superfamily Transporter, Is Required for Sugar Transport, Oxidative Stress Resistance, and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.

Colletotrichum gloeosporioides is the main causal agent of anthracnose in various plant species. Determining the molecular mechanisms underlying the pathogenicity and fungicide resistance of C. gloeosporioides could help build new strategies for disease control. The major facilitator superfamily (MFS) has multiple roles in the transport of a diverse range of substrates. In the present study, an MFS protein CgMFS1 was characterized in C. gloeosporioides. This protein contains seven transmembrane domains, and its predicted 3D structure is highly similar to the reported hexose transporters. To investigate the biological functions of CgMFS1, the gene knock-out mutant ΔCgMFS1 was constructed. A colony growth assay showed that the mutant was remarkably decreased in vegetative growth in minimal medium supplemented with monosaccharides and oligosaccharides as the sole carbon sources, whereas it showed a similar growth rate and colony morphology as wild types when using soluble starch as the carbon source. A stress assay revealed that CgMFS1 is involved in oxidative stress but not in the fungicide resistance of C. gloeosporioides. Furthermore, its pathogenicity was significantly impaired in the mutant, although its appressorium formation was not affected. Our results demonstrate that CgMFS1 is required for sugar transport, resistance to oxidative stress, and the pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.

Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics.

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.

Toward identification of a putative candidate gene for nutrient mineral accumulation in wheat grains for human nutrition purposes.

A multilocus genome-wide association study of a panel of 369 diverse wheat (Triticum aestivum) genotypes was carried out in order to examine the genetic basis of variations in nutrient mineral concentrations in the grains. The panel was grown under field conditions for three consecutive years and the concentrations of Ca, K, Mg, Mn, P, and S were determined. Wide ranges of natural variation were detected among the genotypes. Strong positive correlations were found among the minerals except for K, which showed negative correlation trends with the other minerals. Genetic association analysis detected 86 significant marker-trait associations (MTAs) underlying the natural variations in mineral concentrations in grains. The major MTA was detected on the long arm of chromosome 5A and showed a pleiotropic effect on Ca, K, Mg, Mn, and S. Further significant MTAs were distributed among the whole genome except for chromosomes 3D and 6D. We identified putative candidate genes that are potentially involved in metal uptake, transport, and assimilation, including TraesCS5A02G542600 on chromosome 5A, which was annotated as a Major Facilitator Superfamily transporter and acted on all the minerals except K. TraesCS5A02G542600 was highly expressed in seed coat, and to a lesser extent in the peduncle, awns, and lemma. Our results provide important insights into the genetic basis of enhancement of nutrient mineral concentrations that can help to inform future breeding studies in order to improve human nutrition.

Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform.

The capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.