scholarly journals CFP, the Putative Cercosporin Transporter of Cercospora kikuchii, Is Required for Wild Type Cercosporin Production, Resistance, and Virulence on Soybean

1999 ◽  
Vol 12 (10) ◽  
pp. 901-910 ◽  
Author(s):  
Terrence M. Callahan ◽  
Mark S. Rose ◽  
Maura J. Meade ◽  
Marilyn Ehrenshaft ◽  
Robert G. Upchurch
Keyword(s):  
Major Facilitator Superfamily ◽  
Cdna Clones ◽  
Membrane Transporter ◽  
Wild Type ◽  
Drastic Reduction ◽  
Targeted Disruption ◽  
Reading Frame ◽  
Mrna Transcripts ◽  
Increased Sensitivity ◽  
Major Facilitator

Many species of the fungal genus Cercospora, including the soybean pathogen C. kikuchii, produce the phytotoxic polyketide cercosporin. Cercosporin production is induced by light. Previously, we identified several cDNA clones of mRNA transcripts that exhibited light-enhanced accumulation in C. kikuchii. Targeted disruption of the genomic copy of one of these, now designated CFP (cercosporin facilitator protein), results in a drastic reduction in cercosporin production, greatly reduced virulence of the fungus to soybean, and increased sensitivity to exogenous cercosporin. Sequence analysis of CFP reveals an 1,821-bp open reading frame encoding a 65.4-kDa protein similar to several members of the major facilitator superfamily (MFS) of integral membrane transporter proteins known to confer resistance to various antibiotics and toxins in fungi and bacteria. We propose that CFP encodes a cercosporin transporter that contributes resistance to cercosporin by actively exporting cercosporin, thus maintaining low cellular concentrations of the toxin.

scholarly journals Cysteine Is Exported from theEscherichia coliCytoplasm by CydDC, an ATP-binding Cassette-type Transporter Required for Cytochrome Assembly

2002 ◽  
Vol 277 (51) ◽  
pp. 49841-49849 ◽  
Author(s):  
Marc S. Pittman ◽  
Hazel Corker ◽  
Guanghui Wu ◽  
Marie B. Binet ◽  
Arthur J. G. Moir ◽  
...  
Keyword(s):  
Redox Homeostasis ◽  
Membrane Vesicles ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Major Protein ◽  
Wild Type ◽  
Independent Manner ◽  
Periplasmic Transport ◽  
Major Facilitator ◽  
Atp Binding Cassette

Assembly ofEscherichia colicytochromebdand periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydDmutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in thecydDperiplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropiccydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmicc-type cytochromes incydDand wild-type strains but did not restore cytochromed. Consistent with CydDC being a cysteine exporter,cydDmutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. AcydD orf299double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

scholarly journals Primary familial polycythemia: a frameshift mutation in the erythropoietin receptor gene and increased sensitivity of erythroid progenitors to erythropoietin

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 15-22 ◽  
Author(s):  
L Sokol ◽  
M Luhovy ◽  
Y Guan ◽  
JF Prchal ◽  
GL Semenza ◽  
...  
Keyword(s):  
Receptor Gene ◽  
Erythropoietin Receptor ◽  
Wild Type ◽  
Functional Effect ◽  
Erythroid Progenitors ◽  
Reading Frame ◽  
Increased Sensitivity ◽  
Arterial Po2 ◽  
Mutant Construct ◽  
Serum Erythropoietin

Primary familial and congenital polycythemia (PFCP) is characterized by erythrocytosis with normal arterial PO2, blood P50, and serum erythropoietin (EPO) levels. In two PFCP families EPO receptor (EPOR) polymorphisms cosegregated with PFCP. A heterozygous insertion of G at EPOR nucleotide 5975 was identified in genomic DNA from polycythemic members of family no. 2. 5974insG shifts the reading frame at codon 430, predicting amino acid substitutions and truncation of the last 64 amino acids. Wild-type and mutant EPOR transcripts were detected in erythroid progenitors from affected individuals. Burst-forming units- erythroid from patients exhibited increased colony size and sensitivity to EPO. Transfected Ba/F3 cells expressing EPOR 5974insG exhibited increased EPO sensitivity compared with cells expressing wild-type EPOR. The functional effect of this EPOR mutation was directly compared with the other C-terminal mutations reported in unrelated PFCP families by expression in Ba/F3 cells. The transfected cells with another primary polycythemia associated EPOR mutant construct (G6002A) also exhibited increased sensitivity to EPO.

scholarly journals Bcmfs1, a Novel Major Facilitator Superfamily Transporter from Botrytis cinerea, Provides Tolerance towards the Natural Toxic Compounds Camptothecin and Cercosporin and towards Fungicides

2002 ◽  
Vol 68 (10) ◽  
pp. 4996-5004 ◽  
Author(s):  
Keisuke Hayashi ◽  
Henk-jan Schoonbeek ◽  
Maarten A. De Waard
Keyword(s):  
Botrytis Cinerea ◽  
Abc Transporter ◽  
Pathogenic Fungus ◽  
Major Facilitator Superfamily ◽  
Defense Compounds ◽  
Toxic Compounds ◽  
Major Facilitator Superfamily Transporter ◽  
Dmi Fungicides ◽  
Increased Sensitivity ◽  
Major Facilitator

ABSTRACT 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.

scholarly journals Novel Chromosomally Encoded Multidrug Efflux Transporter MdeA in Staphylococcus aureus

2004 ◽  
Vol 48 (3) ◽  
pp. 909-917 ◽  
Author(s):  
Jianzhong Huang ◽  
Paul W. O'Toole ◽  
Wei Shen ◽  
Heather Amrine-Madsen ◽  
Xinhe Jiang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pathogenic Bacteria ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux ◽  
Expression Library ◽  
Reading Frame ◽  
Transmembrane Segments ◽  
Antibiotic Compounds ◽  
Major Facilitator ◽  
Spontaneous Mutants

ABSTRACT Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

scholarly journals Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus subsp. yingchengensis Strain 10–22

2004 ◽  
Vol 36 (8) ◽  
pp. 519-528 ◽  
Author(s):  
Lei Qin ◽  
Heng-An Wang ◽  
Zhong-Qin Wu ◽  
Xiao-Feng Zhang ◽  
Mei-Lei Jin ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sequence Similarity ◽  
Gene Replacement ◽  
Major Facilitator Superfamily ◽  
Streptomyces Hygroscopicus ◽  
Wild Type ◽  
Gene Encoding ◽  
Major Facilitator

Abstract The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10–22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane α-helical spanners, belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5–20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

scholarly journals Fusarium graminearum Tri12p Influences Virulence to Wheat and Trichothecene Accumulation

2012 ◽  
Vol 25 (11) ◽  
pp. 1408-1418 ◽  
Author(s):  
Jon Menke ◽  
Yanhong Dong ◽  
H. Corby Kistler
Keyword(s):  
Fusarium Graminearum ◽  
Fluorescent Protein ◽  
Toxin Production ◽  
Major Facilitator Superfamily ◽  
Induction Medium ◽  
Wild Type ◽  
In Planta ◽  
Major Facilitator ◽  
Green Fluorescent ◽  
Self Protection

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.

scholarly journals The full-length calcium-sensing receptor dampens the calcemic response to 1α,25(OH)2 vitamin D3 in vivo independently of parathyroid hormone

2009 ◽  
Vol 297 (3) ◽  
pp. F720-F728 ◽  
Author(s):  
Ogo Egbuna ◽  
Steven Quinn ◽  
Lakshmi Kantham ◽  
Robert Butters ◽  
Jiang Pang ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Full Length ◽  
Wild Type ◽  
Calcium Sensing Receptor ◽  
Targeted Disruption ◽  
Deficient Diet ◽  
Calcium Sensing ◽  
Increased Sensitivity ◽  
Key Genes

1α,25(OH)2 vitamin D3 [1,25(OH)2D3] increases serum Ca2+ concentration in vivo, an action counteracted by activation of the Ca2+-sensing receptor (CaSR), which decreases parathyroid hormone (PTH) secretion and increases renal Ca2+ excretion. Relatively little is known of the role the CaSR plays in this response through its potentially direct actions in kidney, gut, and bone independently of PTH. We report PTH-independent roles of the CaSR in modulating the response to exogenous 1,25(OH)2D3 in mice with targeted disruption of both the CaSR and PTH genes (C−P−) compared with that in mice with disruption of the PTH gene alone (C+P−) or wild-type mice (C+P+). After intraperitoneal injection of 0.5 ng/g body wt 1,25(OH)2D3, peak calcemic responses were observed at 24 h in all three genotypes in association with 1) a greater increase in serum Ca2+ in C−P− mice than in the other genotypes on a Ca2+-replete diet that was attenuated by a Ca2+-deficient diet and pamidronate, 2) increased urinary Ca2+-to-creatinine ratios (UCa/Cr) in the C+P− and C+P+ mice but a lowered ratio in the C−P− mice on a Ca2+-replete diet, and 3) no increase in calcitonin (CT) secretion in the C+P+ and C+P− mice and a small increase in the C−P− mice. PTH deficiency had the anticipated effects on the expression of key genes involved in Ca2+ transport at baseline in the duodenum and kidney, and injection of 1,25(OH)2D3 increased gene expression 8 h later. However, the changes in the genes evaluated did not fully explain the differences in serum Ca2+ seen among the genotypes. In conclusion, mice lacking the full-length CaSR have increased sensitivity to the calcemic action of 1,25(OH)2D3 in the setting of PTH deficiency. This is principally from enhanced 1,25(OH)2D3-mediated gut Ca2+ absorption and decreased renal Ca2+ excretion, without any differences in bone-related release of Ca2+ or CT secretion among the three genotypes that could explain the differences in their calcemic responses.

scholarly journals The tfdK Gene Product Facilitates Uptake of 2,4-Dichlorophenoxyacetate by Ralstonia eutrophaJMP134(pJP4)

1998 ◽  
Vol 180 (8) ◽  
pp. 2237-2243 ◽  
Author(s):  
Johan H. J. Leveau ◽  
Alexander J. B. Zehnder ◽  
Jan Roelof van der Meer
Keyword(s):  
Ralstonia Eutropha ◽  
Major Facilitator Superfamily ◽  
Uptake System ◽  
Reading Frame ◽  
Uptake Rates ◽  
Saturation Kinetics ◽  
Uptake Data ◽  
Highly Hydrophobic ◽  
Major Facilitator ◽  
Energy Dependent

ABSTRACT Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Ralstonia eutropha JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 μM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated tfdK, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of thetfdK gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A tfdA mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.

The human iron exporter ferroportin. Insight into the transport mechanism by molecular modeling

2016 ◽  
Vol 12 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Valentina Tortosa ◽  
Maria Carmela Bonaccorsi di Patti ◽  
Giovanni Musci ◽  
Fabio Polticelli
Keyword(s):  
Molecular Modeling ◽  
Transport Mechanism ◽  
Structural Models ◽  
Degradation Pathway ◽  
Atomic Level ◽  
Major Facilitator Superfamily ◽  
Wild Type ◽  
Major Facilitator ◽  
First Time ◽  
Insight Into

AbstractFerroportin, a membrane protein belonging to the major facilitator superfamily of transporters, is the only vertebrate iron exporter known so far. Several ferroportin mutations lead to the so-called ferroportin disease or type 4 hemochromatosis, characterized by two distinct iron accumulation phenotypes depending on whether the mutation affects the activity of the protein or its degradation pathway. Through extensive molecular modeling analyses using the structure of all known major facilitator superfamily members as templates, multiple structural models of ferroportin in the three mechanistically relevant conformations (inward open, occluded, and outward open) have been obtained. The best models, selected on the ground of experimental data available on wild-type and mutant ferroportion, provide for the first time a prediction at the atomic level of the dynamics of the transporter. Based on these results, a possible mechanism for iron export is proposed.

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