The pH-responsive SmrR-SmrT system modulates C. difficile antimicrobial resistance, spore formation, and toxin production.

Clostridioides difficile is an anaerobic gastrointestinal pathogen that spreads through the environment as dormant spores. To survive, replicate, and sporulate in the host intestine, C. difficile must adapt to a variety of conditions in its environment, including changes in pH, the availability of metabolites, host immune factors, and a diverse array of other species. Prior studies showed that changes in intestinal conditions, such as pH, can affect C. difficile toxin production, spore formation, and cell survival. However, little is understood about the specific genes and pathways that facilitate environmental adaptation and lead to changes in C. difficile cell outcomes. In this study, we investigated two genes, CD2505 and CD2506, that are differentially regulated by pH to determine if they impact C. difficile growth and sporulation. Using deletion mutants, we examined the effects of both genes (herein smrR and smrT) on sporulation frequency, toxin production, and antimicrobial resistance. We determined that SmrR is a repressor of smrRT that responds to pH and suppresses sporulation and toxin production through regulation of the SmrT transporter. Further, we showed that SmrT confers resistance to erythromycin and lincomycin, establishing a connection between the regulation of sporulation and antimicrobial resistance.IMPORTANCEClostridioides difficile is a mammalian pathogen that colonizes the large intestine and produces toxins that lead to severe diarrheal disease. C. difficile is a major threat to public health due to its intrinsic resistance to antimicrobials and its ability to form dormant spores that are easily spread from host to host. In this study, we examined the contribution of two genes, smrR and smrT, on sporulation, toxin production, and antimicrobial resistance. Our results indicate that SmrR represses smrT expression, while production of SmrT increases spore and toxin production, as well as resistance to antibiotics.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202.

BACKGROUND: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. METHODS: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. RESULTS: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites. CONCLUSIONS: These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Mutational analysis in Corynebacterium stationis MFS transporters for improving nucleotide bioproduction.

Product secretion from an engineered cell can be advantageous for microbial cell factories. Extensive work on nucleotide manufacturing, one of the most successful microbial fermentation processes, has enabled Corynebacterium stationis to transport nucleotides outside the cell by random mutagenesis; however, the underlying mechanism has not been elucidated, hindering its applications in transporter engineering. Herein, we report the nucleotide-exporting major facilitator superfamily (MFS) transporter from the C. stationis genome and its hyperactive mutation at the G64 residue. Structural estimation and molecular dynamics simulations suggested that the activity of this transporter improved via two mechanisms: (1) enhancing interactions between transmembrane helices through the conserved "RxxQG" motif along with substrate binding and (2) trapping substrate-interacting residue for easier release from the cavity. Our results provide novel insights into how MFS transporters change their conformation from inward- to outward-facing states upon substrate binding to facilitate efflux and can contribute to the development of rational design approaches for efflux improvements in microbial cell factories. KEYPOINTS: • An MFS transporter from C. stationis genome and its mutation at residue G64 were assessed • It enhanced the transporter activity by strengthening transmembrane helix interactions and trapped substrate-interacting residues • Our results contribute to rational design approach development for efflux improvement.

The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola.

Corynespora leaf spot, caused by Corynespora cassiicola, is a foliar disease in cucumber. While the application of quinone outside inhibitors (QoIs) is an effective measure for disease control, it carries the risk of resistance development. In our monitoring of trifloxystrobin resistance from 2008 to 2020, C. cassiicola isolates were categorized into three populations: sensitive isolates (S, 0.01 < EC50 < 0.83 µg/mL), moderately resistant isolates (MR, 1.18 < EC50 < 55.67 µg/mL), and highly resistant isolates (HR, EC50 > 56.98 µg/mL). The resistance frequency reached up to 90% during this period, with an increasing trend observed in the annual average EC50 values of all the isolates. Analysis of the CcCytb gene revealed that both MR and HR populations carried the G143A mutation. Additionally, we identified mitochondrial heterogeneity, with three isolates carrying both G143 and A143 in MR and HR populations. Interestingly, isolates with the G143A mutation (G143A-MR and G143A-HR) displayed differential sensitivity to QoIs. Further experiments involving gene knockout and complementation demonstrated that the major facilitator superfamily (MFS) transporter (CcMfs1) may contribute to the disparity in sensitivity to QoIs between the G143A-MR and G143A-HR populations. However, the difference in sensitivity caused by the CcMfs1 transporter is significantly lower than the differences observed between the two populations. This suggests additional mechanisms contributing to the variation in resistance levels among C. cassiicola isolates. Our study highlights the alarming level of trifloxystrobin resistance in C. cassiicola in China, emphasizing the need for strict prohibition of QoIs use. Furthermore, our findings shed light on the occurrence of both target and non-target resistance mechanisms associated with QoIs in C. cassiicola.

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease.

The melibiose permease of Salmonella typhimurium (MelBSt) catalyzes the stoichiometric symport of galactopyranoside with a cation (H+, Li+, or Na+) and is a prototype for Na+-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelBSt have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelBSt, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na+-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelBSt is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na+-coupled MFS transporters.

Insights into the glycerol transport of Yarrowia lipolytica.

Cellular membranes separate cells from the environment and hence, from molecules essential for their survival. To overcome this hurdle, cells developed specialized transport proteins for the transfer of metabolites across these membranes. Crucial metabolites that need to cross the membrane of each living organism, are the carbon sources. While many organisms prefer glucose as a carbon source, the yeast Yarrowia lipolytica seems to favor glycerol over glucose. The fast growth of Y. lipolytica on glycerol and its flexible metabolism renders this yeast a fascinating organism to study the glycerol metabolism. Based on sequence similarities to the known fungal glycerol transporter ScStl1p and glycerol channel ScFps1p, ten proteins of Y. lipolytica were found that are potentially involved in glycerol uptake. To evaluate, which of these proteins is able to transport glycerol in vivo, a complementation assay with a glycerol transport-deficient strain of Saccharomyces cerevisiae was performed. Six of the ten putative transporters enabled the growth of S. cerevisiae stl1Δ on glycerol and thus, were confirmed as glycerol transporting proteins. Disruption of the transporters in Y. lipolytica abolished its growth on 25 g/L glycerol, but the individual expression of five of the identified glycerol transporters restored growth. Surprisingly, the transporter-disrupted Y. lipolytica strain retained its ability to grow on high glycerol concentrations. This study provides insight into the glycerol uptake of Y. lipolytica at low glycerol concentrations through the characterization of six glycerol transporters and indicates the existence of further mechanisms active at high glycerol concentrations.

Involvement of the SCO3366 efflux pump from S. coelicolor in rifampicin resistance and its regulation by a TetR regulator.

Overexpression of efflux pumps represents a key mechanism of resistance in bacteria. Soil bacteria such as Streptomyces harbour a vast array of efflux genes that are transcriptionally silent under laboratory conditions. However, dissemination of many of these genes into clinical pathogens via horizontal gene transfer results in conferring resistance to multiple drugs. In this study, we have identified the role of a MFS transporter, SCO3366 from Streptomyces coelicolor, in governing multidrug resistance. Overexpression and knockout studies revealed that SCO3366 provides resistance to several structurally unrelated drugs including ciprofloxacin, chloramphenicol, rifampicin and EtBr, with rifampicin being the major substrate. Beyond multidrug resistance, SCO3366 was efficient in providing tolerance towards oxidative stress. A combinatorial mechanism of increased oxidative stress tolerance decreased intracellular drug levels and decreased permeability act synergistically to provide resistance towards rifampicin. Shedding light on the regulation of SCO3366, we find the pump to be directly regulated by the TetR regulator SCO3367 in a negative manner and the repression was found to be relieved in presence of different compounds recognized as substrates of SCO3366. KEY POINTS: • First reported rifampicin efflux pump in Streptomyces coelicolor • Resistance to rifampicin is the result of a synergistic action of increased efflux with increased oxidative stress tolerance and decreased permeability, which can potentially arise in clinically relevant bacteria • SCO3366-SCO3367 to be a novel system that operates to protect the bacteria under varied environmental stress conditions.

Utilization of AlphaFold2 to Predict MFS Protein Conformations after Selective Mutation.

The major facilitator superfamily (MFS) is the largest secondary transporter family and is responsible for transporting a broad range of substrates across the biomembrane. These proteins are involved in a series of conformational changes during substrate transport. To decipher the transport mechanism, it is necessary to obtain structures of these different conformations. At present, great progress has been made in predicting protein structure based on coevolutionary information. In this study, AlphaFold2 was used to predict different conformational structures for 69 MFS transporters of E. coli after the selective mutation of residues at the interface between the N- and C-terminal domains. The predicted structures for these mutants had small RMSD values when compared to structures obtained using X-ray crystallography, which indicates that AlphaFold2 predicts the structure of MSF transporters with high accuracy. In addition, different conformations of other transporter family proteins have been successfully predicted based on mutation methods. This study provides a structural basis to study the transporting mechanism of the MFS transporters and a method to probe dynamic conformation changes of transporter family proteins when performing their function.

A Major Facilitator Superfamily (MFS) Efflux Pump, SCO4121, from Streptomyces coelicolor with Roles in Multidrug Resistance and Oxidative Stress Tolerance and Its Regulation by a MarR Regulator.

Overexpression of efflux pumps is one of the major determinants of resistance in bacteria. Streptomyces species harbor a large array of efflux pumps that are transcriptionally silenced under laboratory conditions. However, their dissemination results in multidrug resistance in different clinical pathogens. In this study, we have identified an efflux pump from Streptomyces coelicolor, SCO4121, belonging to the major facilitator superfamily (MFS) family of transporters and characterized its role in antibiotic resistance. SCO4121 provided resistance to multiple dissimilar drugs upon overexpression in both native and heterologous hosts. Further, deletion of SCO4121 resulted in increased sensitivity toward ciprofloxacin and chloramphenicol, suggesting the pump to be a major transporter of these substrates. Apart from providing multidrug resistance, SCO4121 imparted increased tolerance against the strong oxidant HOCl. In wild-type Streptomyces coelicolor cells, these drugs were found to transcriptionally regulate the pump in a concentration-dependent manner. Additionally, we identified SCO4122, a MarR regulator that positively regulates SCO4121 in response to various drugs and the oxidant HOCl. Thus, through these studies we present the multiple roles of SCO4121 in S. coelicolor and highlight the intricate mechanisms via which it is regulated in response to antibiotics and oxidative stress.IMPORTANCE One of the key mechanisms of drug resistance in bacteria is overexpression of efflux pumps. Streptomyces species are a reservoir of a large number of efflux pumps, potentially to provide resistance to both endogenous and nonendogenous antibiotics. While many of these pumps are not expressed under standard laboratory conditions, they result in resistance to multiple drugs when spread to other bacterial pathogens through horizontal gene transfer. In this study, we have identified a widely conserved efflux pump SCO4121 from Streptomyces coelicolor with roles in both multidrug resistance and oxidative stress tolerance. We also report the presence of an adjacent MarR regulator, SCO4122, which positively regulates SCO4121 in the presence of diverse substrates in a redox-responsive manner. This study highlights that soil bacteria such as Streptomyces can reveal novel mechanisms of antibiotic resistance that may potentially emerge in clinically important bacteria.

Major Facilitator Superfamily Transporter Gene FgMFS1 Is Essential for Fusarium graminearum to Deal with Salicylic Acid Stress and for Its Pathogenicity towards Wheat.

Wheat is a major staple food crop worldwide, due to its total yield and unique processing quality. Its grain yield and quality are threatened by Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum. Salicylic acid (SA) has a strong and toxic effect on F. graminearum and is a hopeful target for sustainable control of FHB. F. graminearum is capable of efficientdealing with SA stress. However, the underlying mechanisms remain unclear. Here, we characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in F. graminearum. FgMFS1 was highly expressed during infection and was upregulated by SA. The predicted three-dimensional structure of the FgMFS1 protein was consistent with the schematic for the antiporter. The subcellular localization experiment indicated that FgMFS1 was usually expressed in the vacuole of hyphae, but was alternatively distributed in the cell membrane under SA treatment, indicating an element of F. graminearum in response to SA. ΔFgMFS1 (loss of function mutant of FgMFS1) showed enhanced sensitivity to SA, less pathogenicity towards wheat, and reduced DON production under SA stress. Re-introduction of a functional FgMFS1 gene into ∆FgMFS1 recovered the mutant phenotypes. Wheat spikes inoculated with ΔFgMFS1 accumulated more SA when compared to those inoculated with the wild-type strain. Ecotopic expression of FgMFS1 in yeast enhanced its tolerance to SA as expected, further demonstrating that FgMFS1 functions as an SA exporter. In conclusion, FgMFS1 encodes an SA exporter in F. graminearum, which is critical for its response to wheat endogenous SA and pathogenicity towards wheat.

Identification of the citrate exporter Cex1 of Yarrowia lipolytica.

Yarrowia lipolytica is a yeast with many talents, one of them being the production of citric acid. Although the citrate biosynthesis is well studied, little is known about the transport mechanism by which citrate is exported. To gain better insight into this mechanism, we set out to identify a transporter involved in citrate export of Y. lipolytica. A total of five proteins were selected for analysis based on their similarity to a known citrate exporter, but neither a citrate transport activity nor any other phenotypic function could be attributed to them. Differential gene expression analysis of two strains with a distinct citrate productivity revealed another three putative transporters, one of which is YALI0D20196p. Disrupting YALI0D20196g in Y. lipolytica abolished citrate production, while extrachromosomal expression enhanced citrate production 5.2-fold in a low producing wildtype. Furthermore, heterologous expression of YALI0D20196p in the non-citrate secreting yeast Saccharomyces cerevisiae facilitated citrate export. Likewise, expression of YALI0D20196p complemented the ability to secrete citrate in an export-deficient strain of Aspergillus niger, confirming a citrate export function of YALI0D20196p. This report on the identification of the first citrate exporter in Y. lipolytica, termed Cex1, represents a valuable starting point for further investigations of the complex transport processes in yeasts.

Genome mining and homologous comparison strategy for digging exporters contributing self-resistance in natamycin-producing Streptomyces strains.

As antibiotics are always toxic to the antibiotic-producing strains themselves, most Streptomyces strains have evolved several self-resistance mechanisms, among which the antibiotic efflux system is understood best and is commonly found. Among the efflux systems, the ATP-binding cassette (ABC) transporter superfamily and the major facilitator superfamily (MFS) are two important transporter families. In this work, the ABC transporters and the MFS transporters from the four reported natamycin-producing Streptomyces strains have been investigated in order to clarify whether these Streptomyces strains share similar efflux strategies for natamycin metabolism. Fifty-one groups of homologous exporter genes were identified as shared by four strains. Differential transcriptional analysis between the natamycin-producing strain Streptomyces chattanoogensis L10 and its ΔscnS0 mutant, which produces no natamycin, reveals that the expression levels of 25 of the above groups of genes were observably changed. The production of natamycin declined over 30% after solely knocking out several of these 25 groups of genes in S. chattanoogensis L10. This indicates that these transporters participate in the efflux of molecules related to natamycin biosynthesis. Our study is the first to demonstrate that the exporters participating in a particular antibiotic metabolism can be excavated and identified quickly by the strategy of genome mining and homologous comparison in the antibiotic-producing strains, leading to deeper understanding of the complex self-resistance mechanisms in Streptomycetes.

Identification and functional characterization of Penicillium marneffei major facilitator superfamily (MFS) transporters.

PENICILLIUM MARNEFFEI: is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H+ antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2, and PmMDR3) were heterologously overexpressed in S. cerevisiae AD∆ conferred resistance to various drugs and compounds including azoles, albeit to different degrees. PmMDR1-expressing strain showed resistance to the broadest range of drugs, followed by the PmMDR3, and PmMDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3, may be able to serve as multidrug efflux pumps.

Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

Towards understanding promiscuity in multidrug efflux pumps.

Drug export from cells is a major factor in the acquisition of cellular resistance to antimicrobial and cancer chemotherapy, and poses a significant threat to future clinical management of disease. Many of the proteins that catalyse drug efflux do so with remarkably low substrate specificity, a phenomenon known as multidrug transport. For these reasons we need a greater understanding of drug recognition and transport in multidrug pumps to inform research that attempts to circumvent their action. Structural and computational studies have been heralded as being great strides towards a full elucidation of multidrug recognition and transport. In this review we summarise these advances and ask how close we are to a molecular understanding of this remarkable phenomenon.

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5'-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6.

The microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. In Sphingobium sp. strain SYK-6, most of the enzyme genes involved in 5,5'-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 (ddvK) and SLG_07780 (ddvR), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A ddvK mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA O-demethylase oxygenase component gene (ligXa), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled ligXa transcriptional regulatory system. A Sphingobium japonicum UT26S transformant expressing ddvK acquired DDVA uptake capacity, indicating that ddvK encodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects of ddvK overexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene (ligI) cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a ddvK-expression plasmid into a ligI mutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives.IMPORTANCE The bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5'-dehydrodivanillate, a lignin-derived biphenyl compound, in Sphingobium sp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5'-dehydrodivanillate.

Identification of the protocatechuate transporter gene in Sphingobium sp. strain SYK-6 and effects of overexpression on production of a value-added metabolite.

Sphingobium sp. strain SYK-6 expresses the best characterized catabolic systems for lignin-derived aromatic compounds. However, the uptake systems for these aromatics remain unknown. In this study, we identified and characterized the protocatechuate (PCA) transporter gene SLG_12880 (pcaK) in SYK-6. Sequence comparisons revealed that PcaK belongs to the aromatic acid/H+ symporter family of major facilitator superfamily transporters. Further, pcaK was highly conserved among Sphingomonadales possessing catabolic genes for vanillate and PCA. The growth of an SYK-6 pcaK mutant was significantly delayed on PCA medium and PCA uptake rate was only 8% of wild type. In addition, vanillate uptake rate was 78% of wild type, although the pcaK mutant grew as well as the wild type on vanillate. When pcaK was expressed in Sphingobium japonicum UT26S, the transformant acquired the capacity to uptake both PCA and vanillate. These results indicate that pcaK is responsible for the major proportion of PCA uptake and a minor fraction of vanillate uptake in SYK-6. The productivity of 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers, was evaluated using a PDC hydrolase SYK-6 mutant harboring a pcaK plasmid. The mutant exhibited 1.27-fold greater PCA conversion and 1.24-fold greater PDC production compared to the control strain, suggesting that enhanced expression of transporter genes for lignin-derived aromatics can be used to increase the production of value-added metabolites.

Membrane-traversing mechanism of thyroid hormone transport by monocarboxylate transporter 8.

Monocarboxylate transporter 8 (MCT8) mediates thyroid hormone (TH) transport across the plasma membrane in many cell types. In order to better understand its mechanism, we have generated three new MCT8 homology models based on sugar transporters XylE in the intracellular opened (PDB ID: 4aj4) and the extracellular partly occluded (PDB ID: 4gby) conformations as well as FucP (PDB ID: 3o7q) and GLUT3 (PDB ID: 4zwc) in the fully extracellular opened conformation. T3-docking studies from both sides revealed interactions with His192, His415, Arg445 and Asp498 as previously identified. Selected mutations revealed further transport-sensitive positions mainly at the discontinuous transmembrane helices TMH7 and 10. Lys418 is potentially involved in neutralising the charge of the TH substrate because it can be replaced by charged, but not by uncharged, amino acids. The side chain of Thr503 was hypothesised to stabilise a helix break at TMH10 that undergoes a prominent local shift during the transport cycle. A T503V mutation accordingly affected transport. The aromatic Tyr419, the polar Ser313 and Ser314 as well as the charged Glu422 and Glu423 lining the transport channel have been studied. Based on related sugar transporters, we suggest an alternating access mechanism for MCT8 involving a series of amino acid positions previously and newly identified as critical for transport.

Sialic acid acquisition in bacteria-one substrate, many transporters.

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, ß-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.

The major facilitator superfamily transporter ZIFL2 modulates cesium and potassium homeostasis in Arabidopsis.

Potassium (K(+)) is an essential mineral nutrient for plant growth and development, with numerous membrane transporters and channels having been implicated in the maintenance and regulation of its homeostasis. The cation cesium (Cs(+)) is toxic for plants but shares similar chemical properties to the K(+) ion and hence competes with its transport. Here, we report that K(+) and Cs(+) homeostasis in Arabidopsis thaliana also requires the action of ZIFL2 (Zinc-Induced Facilitator-Like 2), a member of the Major Facilitator Superfamily (MFS) of membrane transporters. We show that the Arabidopsis ZIFL2 is a functional transporter able to mediate K(+) and Cs(+) influx when heterologously expressed in yeast. Promoter-reporter, reverse transcription-PCR and fluorescent protein fusion experiments indicate that the predominant ZIFL2.1 isoform is targeted to the plasma membrane of endodermal and pericyle root cells. ZIFL2 loss of function and overexpression exacerbate and alleviate plant sensitivity, respectively, upon Cs(+) and excess K(+) supply, also influencing Cs(+) whole-plant partitioning. We propose that the activity of this Arabidopsis MFS carrier promotes cellular K(+) efflux in the root, thereby restricting Cs(+)/K(+) xylem loading and subsequent root to shoot translocation under conditions of Cs(+) or high K(+) external supply.