Major Facilitator Superfamily Gene Research Articles

Brassinosteroid decreases cadmium accumulation via regulating gibberellic acid accumulation and Cd fixation capacity of root cell wall in rice (Oryza sativa)

The precise mechanism behind the association between plants' reactions to cadmium (Cd) stress and brassinosteroid (BR) remains unclear. In the current investigation, Cd stress quickly increased the endogenous BR concentration in the rice roots. Exogenous BR also increased the hemicellulose level in the root cell wall, which in turn increased its capacity to bind Cd. Simultaneously, the transcription level of genes responsible for root Cd absorption was decreased, including Natural Resistance-Associated Macrophage Protein 1/5 (OsNRAMP1/5) and a major facilitator superfamily gene called OsCd1. Ultimately, the increased expression of Heavy Metal ATPase 3 (OsHMA3) and the decreased expression of OsHMA2, which was in charge of separating Cd into vacuoles and translocating Cd to the shoots, respectively, led to a decrease in the amount of Cd that accumulated in the rice shoots. In contrast, transgenic rice lines overexpressing OsGSK2 (a negative regulator in BR signaling) accumulated more Cd, while OsGSK2 RNA interference (RNAi) rice line accumulated less Cd. Furthermore, BR increased endogenous Gibberellic acid (GA) level, and applying GA could replicate its alleviative effect. Taken together, BR decreased Cd accumulation in rice by mediating the cell wall’s fixation capacity to Cd, which might relied on the buildup of the GA.

Auxin is involved in cadmium accumulation in rice through controlling nitric oxide production and the ability of cell walls to bind cadmium

Although auxin has been linked to plants' responses to cadmium (Cd) stress, the exact mechanism is yet elusive. The objective of the current investigation was to determine the role and the mechanism of auxin in controlling rice's Cd accumulation. Rice roots with Cd stress have higher endogenous auxin levels, and exogenous auxin combined Cd treatment could reduce root cell wall's hemicellulose content when compared with Cd treatment alone, which in turn reduced its fixation of Cd, as well as decreased the expression of OsCd1 (a major facilitator superfamily gene), OsNRAMP1/5 (Natural Resistance-Associated Macrophage Protein 1/5), OsZIP5/9 (Zinc Transporter 5/9), and OsHMA2 (Heavy Metal ATPase 2) that participated in Cd uptake and root to shoot translocation. Furthermore, less Cd accumulated in the shoots as a result of auxin's impact in increasing the expression of OsCAL1 (Cadmium accumulation in Leaf 1), OsABCG36/OsPDR9 (G-type ATP-binding cassette transporter/Pleiotropic drug resistance 9), and OsHMA3, which were in charge of Cd efflux and sequestering into vacuoles, respectively. Additionally, auxin decreased endogenous nitric oxide (NO) levels and antioxidant enzyme activity, while treatment of a NO scavenger-cPTIO-reduced auxin's alleviatory effects. In conclusion, the rice's ability to tolerate Cd toxicity was likely increased by the auxin-accelerated cell wall Cd exclusion mechanism, a pathway that controlled by the buildup of NO.

Melatonin reduces cadmium accumulation via mediating the nitric oxide accumulation and increasing the cell wall fixation capacity of cadmium in rice

Melatonin (MT) is participated in plants’ response to cadmium (Cd) tolerance, although its work model remains elusive. Here, the function of MT in adjusting Cd accumulation in rice was investigated. ‘Nipponbare’ (Nip) was cultured in the -Cd (1/2 Kimura B), -Cd + MT (1/2 Kimura B with 1 μM MT), +Cd (1/2 Kimura B plus 1 μM Cd) and +Cd + MT (1/2 Kimura B with 1 μM Cd and 1 μM MT) nutrient solutions for 7 d. Cd markedly induced the endogenous MT accumulation in rice roots and shoots, even within 1 h. MT applied exogenously elevated the hemicelluloses level, which in turn increased the cell wall’s binding capacity to Cd. Furthermore, MT applied exogenously down-regulated the transcription level of Natural Resistance-Associated Macrophage Protein 1 (OsNRAMP1), OsNRAMP5, a major facilitator superfamily gene (OsCd1), and IRON-REGULATED TRANSPORTER 1 (OsIRT1), all of which were responsible for Cd intake, thus less Cd was entered into roots. Moreover, MT applied exogenously also up-regulated transcription level of Cadmium accumulation in Leaf 1 (OsCAL1) and Heavy Metal ATPase 3 (OsHMA3), two genes both attributed to the decreased Cd accumulation in shoots through expelling Cd out of cells and chelating Cd in the vacuoles, respectively. In addition, MT applied exogenously further aggravated the production of nitric oxide (NO) that induced by Cd, while application of a NO donor-SNP mimicked this alleviatory effect of the MT, indicating MT decreased rice Cd accumulation relied on the accumulation of NO.

Detection of major facilitator superfamily (MFS) transporter in Enterobacteriaceae isolated from chicken

A major facilitator superfamily (MFS) transporter is an active secondary transport system that uses a chemiosmotic ion gradient to transport small molecules. MFS is part of the efflux pump group of proteins and is responsible for releasing toxic compounds and contributing to the antimicrobial resistance mechanisms. In this study, we aimed to detect MFS genes from a collection of Enterobacteriaceae (n=60) isolated from cloacal swabs of chickens. After DNA extraction and amplification, only four isolates (6.7%) were regarded positive for MFS genes and subjected to Sanger dideoxy sequencing. A BlastX search in GenBank® showed that four positive samples with approximate length 300 bp were identified as MSF transporter from Klebsiella pneumoniae with a similarity of 97-98%. This finding warrants further study to confirm both phenotypic and genotypic resistance profiles that correlate with bacterial multidrug efflux pump mechanism.

Function of a pathway-associated major facilitator superfamily gene hsaf-orf1 in the biosynthesis of the antifungal HSAF in Lysobacter enzymogenes

HSAF is a potent antifungal natural product with a structure distinct from the antifungal drugs or fungicides in use. The biosynthesis and regulation of HSAF and its analogs have been subject to extensive studies, but the transport of the compounds is still not very clear. In Lysobacter enzymogenes OH11, hsaf-orf1 is the first gene of the HSAF biosynthetic gene cluster (BGC) and has been predicted to encode a major facilitator superfamily (MFS) transporter. We in-frame deleted hsaf-orf1 in strain OH11 and analyzed the metabolite profiles in the total, extracellular and intracellular extracts. The results showed that there was not a significant difference in the distribution of HSAF and its analogs in the ΔORF1 mutant versus in the wild type. Culture media supplemented with glucose or N-acetylglucosamine (GlcNAc) dramatically induced the production of HSAF and analogs in the wild type, but not in ΔORF1. Using fluorescence-labeled glucose 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-d-glucose), we showed that the sugar transport was significantly impaired in ΔORF1. Further analyses by qRT-PCR (quantitative reverse transcription polymerase chain reaction) of a series of key genes for HSAF biosynthesis and regulation supported that hsaf-orf1 is indeed involved in the import of the sugar into OH11. Together, the study unveiled that hsaf-orf1 plays an important role in the regulation and biosynthesis of HSAF and its analogs, through controlling the import of small sugars, which could serve as nutrients as well as signals to induce the antifungal production.

Gibberellic acid alleviates cadmium toxicity in rice by regulating NO accumulation and cell wall fixation capacity of cadmium

Gibberellic acid (GA) has been implicated in the response of plants to cadmium (Cd) stress, but the underlying mechanism remains unclear. In the present study, our aim was to confirm the role of GA in regulating the accumulation of Cd in rice. We found that Cd stress elevated the endogenous GA level in the rice roots. Exogenous GA application not only decreased the fixation of Cd in the root cell wall through reducing the hemicelluloses content, but also decreased the expression of OsNRAMP5 (Natural Resistance-Associated Macrophage Protein 5) and OsCd1 (a major facilitator superfamily gene). Both OsNRAMP5 and OsCd1 are related to Cd absorption, therefore, less Cd was accumulated in the roots. Furthermore, GA increased the expression of OsHMA3 (Heavy Metal ATPase 3) and OsCAL1 (Cadmium accumulation in Leaf 1), which are responsible for sequestering the Cd to the vacuoles and effluxing the Cd outside the cell, respectively, as a result, less Cd was accumulated in the shoots. In contrast, more Cd was accumulated in GA deficient lines. Furthermore, GA decreased the endogenous NO levels and the activity of antioxidant enzymes, while application of a NO scavenger-cPTIO diminished the alleviatory role of GA. In summary, the GA accelerated cell wall Cd exclusion mechanism probably improved rice tolerance to Cd toxicity via regulating the accumulation of NO.

Genome-Wide Analysis of Major Facilitator Superfamily and Its Expression in Response of Poplar to Fusarium oxysporum.

The major facilitator superfamily (MFS) is one of the largest known membrane transporter families. MFSs are involved in many essential functions, but studies on the MFS family in poplar have not yet been reported. Here, we identified 41 MFS genes from Populus trichocarpa (PtrMFSs). We built a phylogenetic tree, which clearly divided members of PtrMFS into six groups with specific gene structures and protein motifs/domains. The promoter regions contain various cis-acting elements involved in stress and hormone responsiveness. Genes derived from segmental duplication events are unevenly distributed in 17 poplar chromosomes. Collinearity analysis showed that PtrMFS genes are conserved and homologous to corresponding genes from four other species. Transcriptome data indicated that 40 poplar MFS genes were differentially expressed when treated with Fusarium oxysporum. Co-expression networks and gene function annotations of MFS genes showed that MFS genes tightly co-regulated and closely related in function of transmembrane transport. Taken together, we systematically analyzed structure and function of genes and proteins in the PtrMFS family. Evidence indicated that poplar MFS genes play key roles in plant development and response to a biological stressor.

The genome of the extremophile Artemia provides insight into strategies to cope with extreme environments

BackgroundBrine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors.Results and discussionHere, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions.ConclusionsA relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.

Constitutive and differential expression of transport protein genes in Parascaris univalens larvae and adult tissues after in vitro exposure to anthelmintic drugs

The equine roundworm Parascaris univalens has developed resistance to the three anthelmintic substances most commonly used in horses. The mechanisms responsible for resistance are believed to be multi-genic, and transport proteins such as the P-glycoprotein (Pgp) family have been suggested to be involved in resistance in several parasites including P. univlaens. To facilitate further research into the mechanisms behind drug metabolism and resistance development in P. univalens we aimed to develop an in vitro model based on larvae. We developed a fast and easy protocol for hatching P. univalens larvae for in vitro studies, resulting in a hatching rate of 92 %. The expression of transport protein genes pgp-2, pgp-9, pgp-11.1, pgp-16.1 and major facilitator superfamily (MFS) genes PgR006_g137 and PgR015_g078 were studied in hatched larvae exposed to the anthelmintic drugs ivermecin (IVM) 10−9 M, pyrantel citrate (PYR) 10−6 M and thiabendazole (TBZ) 10−5 M for 24 h. In comparison, the expression of these transport protein genes was studied in the anterior end and intestinal tissues of adult worms in vitro exposed to IVM, TBZ and PYR, at the same concentrations as larvae, for 3 h, 10 h and 24 h. Larval exposure to sub-lethal doses of IVM for 24 h did not affect the expression levels of any of the investigated genes, however larvae exposed to PYR and TBZ for 24 h showed significantly increased expression of pgp-9. In vitro drug exposure of adult worms did not result in any significant increases in expression of transport protein genes. Comparisons of constitutive expression between larvae and adult worm tissues showed that pgp-9, pgp-11.1, pgp-16.1 and MFS gene PgR015_g078 were expressed at lower levels in larvae than in adult tissues, while pgp-2 and MFS gene PgR006_g137 had similar expression levels in larvae and adult worms. All investigated transport protein genes were expressed at higher rates in the intestine than in the anterior end of adult worms, except pgp-11.1 where the expression was similar between the two tissues. This high constitutive expression in the intestine suggests that this is an important site for xenobiotic efflux in P. univalens. Despite the fact that the results of this study show differences in expression of transport protein genes between larvae and adult tissues, we believe that the larval assay system described here will be an important tool for further research into the molecular mechanisms behind anthelmintic resistance development and for other in vitro studies.

Transcriptomic Analysis of Two Lentinula edodes Genotypes With Different Cadmium Accumulation Ability.

Lentinula edodes, also known as Xiang’gu, is commonly eaten in cultures around the world. However, L. edodes is particularly susceptible to enrichment with heavy metals, particularly cadmium (Cd), which is toxic to human health. Understanding the molecular mechanism and mining key genes involved in Cd enrichment will facilitate genetic modification of L. edodes strains. Two L. edodes genotypes, Le4625 (with higher Cd enrichment capability) and Le4606 (with lower Cd enrichment capability) were used in this study. The Cd concentrations in the mycelia of the tested genotypes differed significantly after Cd (0.1 mg/L) exposure; and the Cd content in Le4625 (1.390 ± 0.098 mg/kg) was approximately three-fold that in Le4606 (0.440 ± 0.038 mg/kg) after 7 h of Cd exposure. A total of 24,592 transcripts were assessed by RNA-Seq to explore variance in Cd accumulation. Firstly, differentially expressed genes (DEGs) were analyzed separately following Cd exposure. In comparison with Ld4625, Ld4606 showed a greater number of Cd-induced changes in transcription. In Ld4606, DEGs following Cd exposure were associated with transmembrane transport, glutathione transfer and cytochrome P450, indicating that these genes could be involved in Cd resistance in L. edodes. Next, Le4606 and Le4625 were exposed to Cd, after which DEGs were identified to explore genetic factors affecting Cd accumulation. After Cd exposure, DEGs between Le4606 and Le4625 encoded proteins involved in multiple biological pathways, including transporters on the membrane, cell wall modification, oxidative stress response, translation, degradation, and signaling pathways. Cadmium enrichment in cells may activate MAPK signaling and the anti-oxidative stress response, which can subsequently alter signal transduction and the intracellular oxidation/reduction balance. Furthermore, several possible candidate genes involved in the Cd accumulation were identified, including the major facilitator superfamily genes, heat shock proteins, and laccase 11, a multicopper oxidase. This comparison of the transcriptomes of two L. edodes strains with different capacities for Cd accumulation provides valuable insight into the cultivation of mushrooms with less Cd enrichment and also serves as a reference for the construction of engineered strains for environmental pollution control.

Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies

Cadmium (Cd) accumulation in rice grain poses a serious threat to human health. While several transport systems have been reported, the complexity of rice Cd transport and accumulation indicates the necessity of identifying additional genes, especially those that are responsible for Cd accumulation divergence between indica and japonica rice subspecies. Here, we show that a gene, OsCd1, belonging to the major facilitator superfamily is involved in root Cd uptake and contributes to grain accumulation in rice. Natural variation in OsCd1 with a missense mutation Val449Asp is responsible for the divergence of rice grain Cd accumulation between indica and japonica. Near-isogenic line tests confirm that the indica variety carrying the japonica allele OsCd1V449 can reduce the grain Cd accumulation. Thus, the japonica allele OsCd1V449 may be useful for reducing grain Cd accumulation of indica rice cultivars through breeding.

Efflux Transporter ArsK Is Responsible for Bacterial Resistance to Arsenite, Antimonite, Trivalent Roxarsone, and Methylarsenite.

Arsenic-resistant bacteria have evolved various efflux systems for arsenic resistance. Five arsenic efflux proteins, ArsB, Acr3, ArsP, ArsJ, and MSF1, have been reported. In this study, comprehensive analyses were performed to study the function of a putative major facilitator superfamily gene, arsK, and the regulation of arsK transcriptional expression in Agrobacterium tumefaciens GW4. We found that (i) arsK is located on an arsenic gene island in strain GW4. ArsK orthologs are widely distributed in arsenic-resistant bacteria and are phylogenetically divergent from the five reported arsenic efflux proteins, indicating that it may be a novel arsenic efflux transporter. (ii) Reporter gene assays showed that the expression of arsK was induced by arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], methylarsenite [MAs(III)], and arsenate [As(V)]. (iii) Heterologous expression of ArsK in an arsenic-hypersensitive Escherichia coli strain showed that ArsK was essential for resistance to As(III), Sb(III), Rox(III), and MAs(III) but not to As(V), dimethylarsenite [dimethyl-As(III)], or Cd(II). (iv) ArsK reduced the cellular accumulation of As(III), Sb(III), Rox(III), and MAs(III) but not to As(V) or dimethyl-As(III). (v) A putative arsenic regulator gene arsR2 was cotranscribed with arsK, and (vi) ArsR2 interacted with the arsR2-arsK promoter region without metalloids and was derepressed by As(III), Sb(III), Rox(III), and MAs(III), indicating the repression activity of ArsR2 for the transcription of arsK These results demonstrate that ArsK is a novel arsenic efflux protein for As(III), Sb(III), Rox(III), and MAs(III) and is regulated by ArsR2. Bacteria use the arsR2-arsK operon for resistance to several trivalent arsenicals or antimonials.IMPORTANCE The metalloid extrusion systems are very important bacterial resistance mechanisms. Each of the previously reported ArsB, Acr3, ArsP, ArsJ, and MSF1 transport proteins conferred only inorganic or organic arsenic/antimony resistance. In contrast, ArsK confers resistance to several inorganic and organic trivalent arsenicals and antimonials. The identification of the novel efflux transporter ArsK enriches our understanding of bacterial resistance to trivalent arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], and methylarsenite [MAs(III)].

RNA-seq for comparative transcript profiling of Phytophthora capsici during its interaction with Arabidopsis thaliana

Phytophthora capsici, a highly dynamic and destructive oomycete pathogen, causes devastating diseases on a wide range of plants worldwide. However, the detailed molecular mechanisms of pathogenicity is still largely unclear. In this study, three different mRNA pool libraries were constructed from its developmental stage, early or late infection stage of the model plant Arabidopsis thaliana, and then were investigated by the RNA-Seq approach. The results demonstrated that 1456 novel transcripts that had not linked to any annotated gene were identified, and 296 genes were found to undergo alternative splicing. Comparative analysis of three different libraries further showed that distinct transcriptional changes of pathogenicity genes were found. A large number of genes containing cell wall degrading enzymes, major facilitator superfamily genes and cytochrome P450 genes were highly induced during infection. In addition, several types of well-known effectors including RxLR, CRN, Elicitin and NLP proteins also showed high transcript abundances during infection. The transcriptional levels of six effector genes during the infection process were further validated by qRT-PCR. Collectively, this study provides a basic understanding of pathogenic mechanisms of P. capsici during the interaction with plants.

The mycoparasitic fungus Clonostachys rosea responds with both common and specific gene expression during interspecific interactions with fungal prey.

Clonostachys rosea is a necrotrophic mycoparasitic fungus, used for biological control of plant pathogenic fungi. A better understanding of the underlying mechanisms resulting in successful biocontrol is important for knowledge‐based improvements of the application and use of biocontrol in agricultural production systems. Transcriptomic analyses revealed that C. rosea responded with both common and specific gene expression during interactions with the fungal prey species Botrytis cinerea and Fusarium graminearum. Genes predicted to encode proteins involved in membrane transport, biosynthesis of secondary metabolites and carbohydrate‐active enzymes were induced during the mycoparasitic attack. Predicted major facilitator superfamily (MFS) transporters constituted 54% of the induced genes, and detailed phylogenetic and evolutionary analyses showed that a majority of these genes belonged to MFS gene families evolving under selection for increased paralog numbers, with predicted functions in drug resistance and transport of carbohydrates and small organic compounds. Sequence analysis of MFS transporters from family 2.A.1.3.65 identified rapidly evolving loop regions forming the entry to the transport tunnel, indicating changes in substrate specificity as a target for selection. Deletion of the MFS transporter gene mfs464 resulted in mutants with increased growth inhibitory activity against F. graminearum, providing evidence for a function in interspecific fungal interactions. In summary, we show that the mycoparasite C. rosea can distinguish between fungal prey species and modulate its transcriptomic responses accordingly. Gene expression data emphasize the importance of secondary metabolites in mycoparasitic interactions.

Stachyose is a preferential carbon source utilized by the rice false smut pathogen, Villosiclava virens

Villosiclava virens is the causal pathogen of rice false smut (RFS) disease. RFS is an emerging panicle disease, concerning rice production worldwide. However, the biology and pathogenicity of V. virens are still not well-understood. In this study, we investigated the sugar preference of V. virens and the underlying mechanism on how the favored sugar promoted the fungal growth. The results demonstrated that V. virens preferentially utilized stachyose over sucrose that is previously reported to be the best carbon source for V. virens. Stachyose could promote conidia germination, hyphae elongation and mycelium growth of V. virens. Transcriptome and qRT-PCR analyses showed that genes involved in transporting carbohydrate, amino acid, inorganic ion and secondary metabolites were mostly up-regulated by stachyose than by sucrose. Moreover, Uv8b_6977 encoding a putative major facilitator superfamily (MFS) transporter was highly and specifically induced by stachyose, indicating that it may be a stachyose transporter. Expression levels of several stachyose-inducible MFS genes were also highly induced in V. virens-infected rice spikelets especially when early false smut balls appeared, implicating that these genes may play important roles in the formation of false smut balls. Collectively, this work identifies stachyose as a preferential carbon source for V. virens, and provides gene candidates for investigation of the sugar utilization mechanism of V. virens.

MFS transportome of the human pathogenic yeast Candida albicans

BackgroundThe major facilitator superfamily (MFS) is one of the two largest superfamilies of membrane transporters present ubiquitously in bacteria, archaea, and eukarya and includes members that function as uniporters, symporters or antiporters. We report here the complete transportome of MFS proteins of a human pathogenic yeast Candida albicans.ResultsComputational analysis of C. albicans genome enabled us to identify 95 potential MFS proteins which clustered into 17 families using Saier's Transport Commission (TC) system. Among these SP, DHA1, DHA2 and ACS represented major families consisting of 22, 22, 9 and 16 members, respectively. Family designations in C. albicans were validated by subjecting Saccharomyces cerevisiae genome to TC system. Based on the published available genomics/proteomics data, 87 of the putative MFS genes of C. albicans were found to express either at mRNA or protein levels. We checked the expression of the remaining 8 genes by using RT-PCR and observed that they are not expressed under basal growth conditions implying that either these 8 genes are expressed under specific growth conditions or they may be candidates for pseudogenes.ConclusionThe in silico characterisation of MFS transporters in Candida albicans genome revealed a large complement of MFS transporters with most of them showing expression. Considering the clinical relevance of C. albicans and role of MFS members in antifungal resistance and nutrient transport, this analysis would pave way for identifying their physiological relevance.

Functional Analysis of ABC Transporter Genes FromBotrytis cinereaIdentifies BcatrB as a Transporter of Eugenol

The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expression of 11 ABC (BcatrA-BcatrK) and three MFS genes (Bcmfs1, Bcmfs2 and Bcmfs4) was studied. All genes showed a low basal level of expression, but were differentially induced by treatment with cycloheximide and the plant defence compounds camptothecin, eugenol, psoralen, resveratrol and rishitin. The latter compounds induced expression of BcatrB at a high level. Eugenol was more toxic to BcatrB gene-replacement mutants than to the control isolates. Eugenol also caused an instantaneous increase in mycelial accumulation of the fungicide fludioxonil, a known substrate of BcatrB. However, there was no difference in virulence between the wild-type and BcatrB gene-replacement mutants on Ocimum basilicum, a plant known to contain eugenol. The results indicate that BcatrB is a transporter of lipophilic compounds, such as eugenol, but its role in virulence remains uncertain.

A semiautomated approach to gene discovery through expressed sequence tag data mining: discovery of new human transporter genes.

Identification and functional characterization of the genes in the human genome remain a major challenge. A principal source of publicly available information used for this purpose is the National Center for Biotechnology Information database of expressed sequence tags (dbEST), which contains over 4 million human ESTs. To extract the information buried in this data more effectively, we have developed a semiautomated method to mine dbEST for uncharacterized human genes. Starting with a single protein input sequence, a family of related proteins from all species is compiled. This entire family is then used to mine the human EST database for new gene candidates. Evaluation of putative new gene candidates in the context of a family of characterized proteins provides a framework for inference of the structure and function of the new genes. When applied to a test data set of 28 families within the major facilitator superfamily (MFS) of membrane transporters, our protocol found 73 previously characterized human MFS genes and 43 new MFS gene candidates. Development of this approach provided insights into the problems and pitfalls of automated data mining using public databases.

Common Features in Substrates of Multidrug Resistance Transporters

Multidrug resistance (MDR) proteins serve as transporters for chemical compounds, small molecules like antibiotics in almost all species from bacteria to higher eukaryote. They provide the resistance against antibiotics and chemicals for the cells like cancer, fungi and parasite, etc. Therefore, the research on MDR proteins is essential and important in medical, agricultural and scientific fields. Five transporter families are known to contain MDR members as follows. ABC (ATP-binding cassette superfamily), MFS (major-facilitator superfamily), SMR (small multidrug resistance family), RND (resistance-nodulation cell division family), and MATE (multidrug and toxic compound extrusion family). The ABC transporter consists of some subfamilies, such as pgp, mrp and pdr. The ABC transporter is driven by ATP hydrolysis energy, and the other four families are driven by proton/sodium motive force. It was reported that more than 90% of MDR genes are composed of ABC and MFS genes in completely sequenced bacteria genomes [4]. In general, the relationship of the transporter and its substrate is highly specific, but MDRs are known to be transporters that have broad spectrums of drugs and chemical compounds. Thus, MDRs are poly-specific rather than nonspecific transporters [5]. Many substrates have been found and used in experiments for characterizing properties of MDRs. The spectrum of each MDR family is known to be somewhat distinct, but the relationship to the structural and physicochemical properties of substrates has not been well established. Previously, we collected chemical compound structures for substrates of MDR proteins from the published literature and tried to retrieve structural and physicochemical properties [2], They were represented by 67 kinds of the KEGG atom types [1]. In this study, we classified these collected chemical compound substrates and extracted common sub-graphs of them by using Vzyme [3] in order to detect putative recognition sites in chemical compound substrates by the MDR protein.

Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides.

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.