Toxic Compound Extrusion Transporters Research Articles

Metagenomic profiles of the antimicrobial resistance in traditional Chinese fermented meat products: Core resistome and co-occurrence patterns

Antimicrobial resistance (AMR) poses a significant challenge to global health, and the presence of antibiotic resistance genes (ARGs) in food poses a potential threat to public health. Traditional Chinese fermented meat products (FMPs) are highly favored because of their unique flavors and cultural value. However, microbial safety and the potential distribution and composition of AMR in these products remain unclear. In this study, a comprehensive analysis of bacterial composition and antibiotic-resistant populations in 216 samples of traditional fermented meat products from different regions of China was conducted using a metagenomic approach. Staphylococcus was the most abundant genus in the samples, accounting for an average abundance of 29.9 %, followed by Tetragenococcus (17.1 %), and Latilactobacillus (3.6 %). A core resistome of FMP samples was constructed for the first time using co-occurrence network analysis, which revealed the distribution and interrelationships of ARGs and bio/metal-resistant genes (BMRGs). Random forest analysis identified the lincosamide nucleotidyltransferase lnuA and the multidrug and toxic compound extrusion (MATE) transporter abeM as potential indicators for assessing the overall abundance of the core resistome. Additionally, Staphylococcus, Acinetobacter, and Pseudomonas were identified as hosts constituting the core resistome. Despite their low abundance, the latter two still serve as major reservoirs of antibiotic resistance genes. Notably, Lactococcus cremoris was identified as the key host for tetracycline resistance genes in the samples, highlighting the need for enhanced resistance monitoring in lactic acid bacteria. Based on our findings, in the microbial safety assessment of fermented meat products, beyond common foodborne pathogens, attention should be focused on detecting and controlling coagulase-negative Staphylococcus, Acinetobacter, and Pseudomonas, and addressing bacterial resistance. The quantitative detection of lnuA and abeM could provide a convenient and rapid method for assessing the overall abundance of the core resistome. Our findings have important implications for the control of bacterial resistance and prevention of pathogenic bacteria in fermented meat products.

GmMATE100 Is Involved in the Import of Soyasaponins A and B into Vacuoles in Soybean Plants (Glycine max L.).

Triterpenoid saponins, synthesized via the mevalonic acid (MVA) pathway in the cytoplasm, provide protection against pathogens and pests in plants and health benefits for humans. However, the mechanisms by which triterpenoid saponins are transported between cellular compartments remain uncharacterized. Here, we characterize a tonoplast localized multidrug and toxic compound extrusion transporter, GmMATE100 (encoded by Glyma.18G143700), from soybean (Glycine max L.). GmMATE100 is co-expressed with soyasaponin biosynthetic genes, and its expression was induced by MeJA treatment, which also led to soyasaponin accumulation in soybean roots. GmMATE100 efficiently transports multiple type-B soyasaponins as well as type-A soyasaponins with low affinity from the cytosol to the vacuole in a yeast system. The GmMATE100 loss-of-function mutant showed a significant decrease in type-A and type-B soyasaponin contents in soybean roots. This study not only characterized the first soybean triterpenoid saponin transporter but also provided new knowledge for the rational engineering of soyasaponin content and composition in soybean plants to modulate their levels within crop environments.

A CYP78As-small grain4-coat protein complex Ⅱ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

A citrate efflux transporter important for manganese distribution and phosphorus uptake in rice.

The plant citrate transporters, functional in mineral nutrient uptake and homeostasis, usually belong to the multidrug and toxic compound extrusion transporter family. We identified and functionally characterized a rice (Oryza sativa) citrate transporter, OsCT1, which differs from known plant citrate transporters and is structurally close to rice silicon transporters. Domain analysis depicted that OsCT1 carries a bacterial citrate-metal transporter domain, CitMHS. OsCT1 showed citrate efflux activity when expressed in Xenopus laevis oocytes and is localized to the cell plasma membrane. It is highly expressed in the shoot and reproductive tissues of rice, and its promoter activity was visible in cells surrounding the vasculature. The OsCT1 knockout (KO) lines showed a reduced citrate content in the shoots and the root exudates, whereas overexpression (OE) line showed higher citrate exudation from their roots. Further, the KO and OE lines showed variations in the manganese (Mn) distribution leading to changes in their agronomical traits. Under deficient conditions (Mn-sufficient conditions followed by 8 days of 0 μm MnCl2 · 4H2 O treatment), the supply of manganese towards the newer leaf was found to be obstructed in the KO line. There were no significant differences in phosphorus (P) distribution; however, P uptake was reduced in the KO and increased in OE lines at the vegetative stage. Further, experiments in Xenopus oocytes revealed that OsCT1 could efflux citrate with Mn. In this way, we provide insights into a mechanism of citrate-metal transport in plants and its role in mineral homeostasis, which remains conserved with their bacterial counterparts.

Functional identification of PhMATE1 in flower color formation in petunia.

Multidrug and toxic compound extrusion (MATE) transporter proteins are a class of secondary transporter proteins that can transport flavonoids. Anthocyanins, a kind of flavonoid, are important secondary metabolites widely found in higher plants; they determine the flower color of most angiosperms. TT12 in Arabidopsis was the first MATE protein identified to be involved in flavonoid transport. Petunia (Petunia hybrida) is an important ornamental plant and is one of the ideal plants for studying plant flower color. However, there are few reports on anthocyanin transport in petunia. In this study, we characterized a homolog of Arabidopsis TT12 in the petunia genome, PhMATE1, that shares the highest amino acid sequence identity with Arabidopsis TT12. PhMATE1 protein contained 11 transmembrane helices. PhMATE1 showed a high transcription level in corollas. The silencing of PhMATE1 mediated by both virus-induced gene silence and RNA interference changed flower color and reduced anthocyanin content in petunia, suggesting that PhMATE1 is involved in anthocyanin transport in petunia. Furthermore, PhMATE1 silencing downregulated the expression of the structural genes of the anthocyanin synthesis pathway. The results of this study supported the hypothesis that MATEs are involved in the sequestration of anthocyanins during flower color formation.

Genome-wide identification of MATE, functional analysis and molecular dynamics of DcMATE21 involved in anthocyanin accumulation in Daucus carota

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs, DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota.

Global analysis of common bean multidrug and toxic compound extrusion transporters (PvMATEs): PvMATE8 and pinto bean seed coat darkening.

In common bean (Phaseolus vulgaris L.), postharvest seed coat darkening is an undesirable trait that affects crop value. The increased accumulation of proanthocyanidins (PAs) in the seed coat results in darker seeds in many market classes of colored beans after harvest. The precursors of PAs are synthesized in the cytoplasm, and subsequently get glycosylated and then transported to the vacuoles where polymerization occurs. Thus, vacuolar transporters play an important role in the accumulation of PAs. Here, we report that common bean genome contains 59 multidrug and toxic compound extrusion genes (PvMATEs). Phylogenetic analysis of putative PvMATEs with functionally characterized MATEs from other plant species categorized them into substrate-specific clades. Our data demonstrate that a vacuolar transporter PvMATE8 is expressed at a higher level in the pinto bean cultivar CDC Pintium (regular darkening) compared to 1533-15 (slow darkening). PvMATE8 localizes in the vacuolar membrane and rescues the PA deficient (tt12) mutant phenotype in Arabidopsis thaliana. Analysis of PA monomers in transgenic seeds together with wild-type and mutants suggests a possible feedback regulation of PA biosynthesis and accumulation. Identification of PvMATE8 will help better understand the mechanism of PA accumulation in common bean.

Biochemical analysis of anthocyanin and proanthocyanidin and their regulation in determining chickpea flower and seed coat colour.

Flower and seed coat colour are important agronomic traits in chickpea (Cicer arietinum L.). Cultivated chickpeas are of two types namely, desi (dark seeded, purple flowered) and kabuli (light seeded, white flowered). There has been limited information about the molecular mechanism underlying colour variation of flower and seed coats in desi and kabuli chickpea. We profiled the anthocyanin and proanthocyanidin (PA) contents in chickpea flowers and seed coats. Tissue-specific silencing of two genes encoding a basic helix-loop-helix (CabHLH) protein and a tonoplast-localized multidrug and toxic compound extrusion (CaMATE1) transporter in a desi genotype resulted in the reduction in expression of anthocyanin and PA biosynthetic genes and anthocyanin and PA contents in the flower and seed coat, and produced flowers and seeds with kabuli characteristics. Transcriptional regulation of a subset of anthocyanin and PA biosynthetic genes by a natural CabHLH variant and transport assay of a natural CaMATE1 variant explained the association of these alleles with the kabuli phenotype. We carried out a detailed molecular characterization of these genes, and provided evidence that kabuli chickpea flower and seed colour phenotypes can be derived by manipulation of single genes in a desi chickpea background.

Identification of two chickpea multidrug and toxic compound extrusion transporter genes transcriptionally upregulated upon aluminum treatment in root tips.

Aluminum (Al) toxicity poses a significant challenge for the yield improvement of chickpea, which is an economically important legume crop with high nutritional value in human diets. The genetic basis of Al-tolerance in chickpea remains unclear. Here, we assessed the Al-tolerance of 8 wild Cicer and one cultivated chickpea (PBA Pistol) accessions by measuring the root elongation in solution culture under control (0 μM Al3+) and Al treatments (15, 30 μM Al3+). Compared to PBA Pistol, the wild Cicer accessions displayed both tolerant and sensitive phenotypes, supporting wild Cicer as a potential genetic pool for Al-tolerance improvement. To identify potential genes related to Al-tolerance in chickpea, genome-wide screening of multidrug and toxic compound extrusion (MATE) encoding genes was performed. Fifty-six MATE genes were identified in total, which can be divided into 4 major phylogenetic groups. Four chickpea MATE genes (CaMATE1-4) were clustered with the previously characterized citrate transporters MtMATE66 and MtMATE69 in Medicago truncatula. Transcriptome data showed that CaMATE1-4 have diverse expression profiles, with CaMATE2 being root-specific. qRT-PCR analyses confirmed that CaMATE2 and CaMATE4 were highly expressed in root tips and were up-regulated upon Al treatment in all chickpea lines. Further measurement of carboxylic acids showed that malonic acid, instead of malate or citrate, is the major extruded acid by Cicer spp. root. Protein structural modeling analyses revealed that CaMATE2 has a divergent substrate-binding cavity from Arabidopsis AtFRD3, which may explain the different acid-secretion profile for chickpea. Pangenome survey showed that CaMATE1-4 have much higher genetic diversity in wild Cicer than that in cultivated chickpea. This first identification of CaMATE2 and CaMATE4 responsive to Al3+ treatment in Cicer paves the way for future functional characterization of MATE genes in Cicer spp., and to facilitate future design of gene-specific markers for Al-tolerant line selection in chickpea breeding programs.

Genome-Wide Analysis of Multidrug and Toxic Compound Extruction Transporters in Grape.

Grape (Vitis vinifera L.) is an important fruit crop in the world. It is used as a table grape and is also used for raisin and wine production. Grape berries accumulate secondary metabolites, such as anthocyanins, tannins, and resveratrol, which are known as functional compounds for human health. Multidrug and toxic compound extrusion transporter (MATEs) transport secondary metabolites. MATEs also transport other solutes, including organic acids, and toxic xenobiotics, depending on cation gradient and play various roles in plants. MATE comprises 300–500 amino acid residues and possesses a MATE domain and 8–12 transmembrane domains. In the present study, 59 MATE genes were identified in the grape genome, and phylogenetic analysis revealed the presence of four groups of grape MATEs (Group 1–4). Their information, such as gene structures, protein motifs, predicted subcellular localizations, and gene IDs of four genome annotations, that is, CRIBI v1, CRIBI v2, Genoscope, and Vcost v3, were annotated. The transport substrates and physiological functions of grape MATEs were estimated based on their homology with the analyzed MATEs in other plant species. Group 1 may transport toxic compounds and alkaloids, Group 2 may transport polyphenolic compounds, Group 3 may transport organic acids, and Group 4 may transport plant hormones related to signal transduction. In addition to the known anthocyanin transporters, VvMATE37 and VvMATE39, a novel anthocyanin transporter, VvMATE38 in Group 2, was suggested as a key transporter for anthocyanin accumulation in grape berry skin. VvMATE46, VvMATE47, and VvMATE49 in Group 3 may contribute to Al3+ detoxification and Fe2+/Fe3+ translocation via organic acid transport. This study provides helpful and fundamental information for grape MATE studies and resolves the confusion of gene IDs in different genome annotations.

Transport engineering using tobacco transporter NtJAT1 enhances alkaloid production in Escherichia coli.

Transporters have been used in the production of plant metabolites in microorganisms. This study introduced a tobacco multidrug and toxic compound extrusion transporter, NtJAT1, into alkaloid-producing Escherichia coli cells. NtJAT1 expression enhanced alkaloid production secretion into the medium by 14 folds. Our findings further demonstrate the usefulness of the transport-engineering approach.

The Roles of Multidrug and Toxic Compound Extrusion (MATE) Transporters in Regulating Agronomic Traits

Multidrug and toxic compound extrusion (MATE) transporters are ancient proteins conserved among various kingdoms, from prokaryotes to eukaryotes. In plants, MATEs usually form a large family in the genome. Homologous MATE transporters have different subcellular localizations, substrate specificities, and responses to external stimuli for functional differentiations. The substrates of MATEs in plants include polyphenols, alkaloids, phytohormones, and ion chelators. The accumulation of these substrates is often associated with favorable agronomic traits such as seed and fruit colors, the balance between dormancy and germination, taste, and stress adaptability. In crops, wild germplasms and domesticated germplasms usually have contrasting agronomic traits such as seed color, seed taste, and stress tolerance. MATE transporters are involved in the regulations of these traits. In this review, we discuss the uniqueness and significance of there being such a large family of MATEs in plants, their substrate diversity that enables them to be involved in various agronomic traits, and the allelic forms and the expression patterns of MATE that are associated with favorable agronomic traits in domesticated crops. The understanding on the roles of MATEs in regulating favorable agronomic traits in crops will provide hints for the selection of genes for molecular breeding that improve desirable traits.

High Resolution Genome Wide Association Studies Reveal Rich Genetic Architectures of Grain Zinc and Iron in Common Wheat (Triticum aestivum L.).

Biofortification is a sustainable strategy to alleviate micronutrient deficiency in humans. It is necessary to improve grain zinc (GZnC) and iron concentrations (GFeC) in wheat based on genetic knowledge. However, the precise dissection of the genetic architecture underlying GZnC and GFeC remains challenging. In this study, high-resolution genome-wide association studies were conducted for GZnC and GFeC by three different models using 166 wheat cultivars and 373,106 polymorphic markers from the wheat 660K and 90K single nucleotide polymorphism (SNP) arrays. Totally, 25 and 16 stable loci were detected for GZnC and GFeC, respectively. Among them, 17 loci for GZnC and 8 for GFeC are likely to be new quantitative trait locus/loci (QTL). Based on gene annotations and expression profiles, 28 promising candidate genes were identified for Zn/Fe uptake (8), transport (11), storage (3), and regulations (6). Of them, 11 genes were putative wheat orthologs of known Arabidopsis and rice genes related to Zn/Fe homeostasis. A brief model, such as genes related to Zn/Fe homeostasis from root uptake, xylem transport to the final seed storage was proposed in wheat. Kompetitive allele-specific PCR (KASP) markers were successfully developed for two major QTL of GZnC on chromosome arms 3AL and 7AL, respectively, which were independent of thousand kernel weight and plant height. The 3AL QTL was further validated in a bi-parental population under multi-environments. A wheat multidrug and toxic compound extrusion (MATE) transporter TraesCS3A01G499300, the ortholog of rice gene OsPEZ2, was identified as a potential candidate gene. This study has advanced our knowledge of the genetic basis underlying GZnC and GFeC in wheat and provides valuable markers and candidate genes for wheat biofortification.

A Systematic Phylogenomic Classification of the Multidrug and Toxic Compound Extrusion Transporter Gene Family in Plants.

Multidrug and toxic compound extrusion (MATE) transporters comprise a multigene family that mediates multiple functions in plants through the efflux of diverse substrates including organic molecules, specialized metabolites, hormones, and xenobiotics. MATE classification based on genome-wide studies remains ambiguous, likely due to a lack of large-scale phylogenomic studies and/or reference sequence datasets. To resolve this, we established a phylogeny of the plant MATE gene family using a comprehensive kingdom-wide phylogenomic analysis of 74 diverse plant species. We identified more than 4,000 MATEs, which were classified into 14 subgroups based on a systematic bioinformatics pipeline using USEARCH, blast+ and synteny network tools. Our classification was performed using a four-step process, whereby MATEs sharing ≥ 60% protein sequence identity with a ≤ 1E-05 threshold at different sequence lengths (either full-length, ≥ 60% length, or ≥ 150 amino acids) or retaining in the similar synteny blocks were assigned to the same subgroup. In this way, we assigned subgroups to 95.8% of the identified MATEs, which we substantiated using synteny network clustering analysis. The subgroups were clustered under four major phylogenetic groups and named according to their clockwise appearance within each group. We then generated a reference sequence dataset, the usefulness of which was demonstrated in the classification of MATEs in additional species not included in the original analysis. Approximately 74% of the plant MATEs exhibited synteny relationships with angiosperm-wide or lineage-, order/family-, and species-specific conservation. Most subgroups evolved independently, and their distinct evolutionary trends were likely associated with the development of functional novelties or the maintenance of conserved functions. Together with the systematic classification and synteny network profiling analyses, we identified all the major evolutionary events experienced by the MATE gene family in plants. We believe that our findings and the reference dataset provide a valuable resource to guide future functional studies aiming to explore the key roles of MATEs in different aspects of plant physiology. Our classification framework can also be readily extendable to other (super) families.

The Tiny Companion Matters: The Important Role of Protons in Active Transports in Plants.

In plants, the translocation of molecules, such as ions, metabolites, and hormones, between different subcellular compartments or different cells is achieved by transmembrane transporters, which play important roles in growth, development, and adaptation to the environment. To facilitate transport in a specific direction, active transporters that can translocate their substrates against the concentration gradient are needed. Examples of major active transporters in plants include ATP-binding cassette (ABC) transporters, multidrug and toxic compound extrusion (MATE) transporters, monosaccharide transporters (MSTs), sucrose transporters (SUTs), and amino acid transporters. Transport via ABC transporters is driven by ATP. The electrochemical gradient across the membrane energizes these secondary transporters. The pH in each cell and subcellular compartment is tightly regulated and yet highly dynamic, especially when under stress. Here, the effects of cellular and subcellular pH on the activities of ABC transporters, MATE transporters, MSTs, SUTs, and amino acid transporters will be discussed to enhance our understanding of their mechanics. The relation of the altered transporter activities to various biological processes of plants will also be addressed. Although most molecular transport research has focused on the substrate, the role of protons, the tiny counterparts of the substrate, should also not be ignored.

Identifying Quantitative Trait Loci and Candidate Genes Conferring Resistance to Soybean Mosaic Virus SC7 by Quantitative Trait Loci-Sequencing in Soybean.

Soybean mosaic virus (SMV) is detrimental to soybean (Glycine max) breeding, seed quality, and yield worldwide. Improving the basic resistance of host plants is the most effective and economical method to reduce damage from SMV. Therefore, it is necessary to identify and clone novel SMV resistance genes. Here, we report the characterization of two soybean cultivars, DN50 and XQD, with different levels of resistance to SMV. Compared with XQD, DN50 exhibits enhanced resistance to the SMV strain SC7. By combining bulked-segregant analysis (BSA)-seq and fine-mapping, we identified a novel resistance locus, RSMV-11, spanning an approximately 207-kb region on chromosome 11 and containing 25 annotated genes in the reference Williams 82 genome. Of these genes, we identified eleven with non-synonymous single-nucleotide polymorphisms (SNPs) or insertion-deletion mutations (InDels) in their coding regions between two parents. One gene, GmMATE68 (Glyma.11G028900), harbored a frameshift mutation. GmMATE68 encodes a multidrug and toxic compound extrusion (MATE) transporter that is expressed in all soybean tissues and is induced by SC7. Given that MATE transporter families have been reported to be linked with plant disease resistance, we suggest that GmMATE68 is responsible for SC7 resistance in DN50. Our results reveal a novel SMV-resistance locus, improving understanding of the genetics of soybean disease resistance and providing a potential new tool for marker-assisted selection breeding in soybean.

The Poly-Glutamate Motif of GmMATE4 Regulates Its Isoflavone Transport Activity.

Multidrug and toxic compound extrusion (MATE) transporters in eukaryotes have been characterized to be antiporters that mediate the transport of substrates in exchange for protons. In plants, alkaloids, phytohormones, ion chelators, and flavonoids have been reported to be the substrates of MATE transporters. Structural analyses have been conducted to dissect the functional significance of various motifs of MATE proteins. However, an understanding of the functions of the N- and C-termini has been inadequate. Here, by performing phylogenetic analyses and protein sequence alignment of 14 representative plant species, we identified a distinctive N-terminal poly-glutamate motif among a cluster of MATE proteins in soybean. Amongst them, GmMATE4 has the most consecutive glutamate residues at the N-terminus. A subcellular localization study showed that GmMATE4 was localized at the vacuolar membrane-like structure. Protein charge prediction showed that the mutation of the glutamate residues to alanine would reduce the negative charge at the N-terminus. Using yeast as the model, we showed that GmMATE4 mediated the transport of daidzein, genistein, glycitein, and glycitin. In addition, the glutamate-to-alanine mutation reduced the isoflavone transport capacity of GmMATE4. Altogether, we demonstrated GmMATE4 as an isoflavone transporter and the functional significance of the N-terminal poly-glutamate motif of GmMATE4 for regulating the isoflavone transport activity.

Alternating access switching in molecular dynamics simulations of PfMATE transporter

The multidrug and toxic compound extrusion (MATE) transporters expel chemical compounds out of the cell which results in multidrug resistance. Recently, a distinct conformation of the inward-facing crystal structures for DinF protein from Pyrococcus furiosus (PfMATE) has been determined [Zakrzewska et al. (1)]. This has allowed investigations of the mechanisms of conformational transitions between inward- and outward-facing structures of the PfMATE. We performed molecular dynamics simulations of the inward-facing (IF) and the outward-facing (OF) PfMATE in the native archaeal lipid bilayer to investigate the atomistic level details of the conformational changes.

The structure of the Aquifex aeolicus MATE family multidrug resistance transporter and sequence comparisons suggest the existence of a new subfamily

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H+ or Na+ to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-Å resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family.

Transport engineering for improving the production and secretion of valuable alkaloids in Escherichia coli

Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing Escherichia coli strain into which the multidrug and toxic compound extrusion transporter Arabidopsis AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.