The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2443-2450 ◽  
Author(s):  
Abdelhamid Abbas ◽  
John E. McGuire ◽  
Delores Crowley ◽  
Christine Baysse ◽  
Max Dow ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Stress Tolerance ◽  
Major Facilitator Superfamily ◽  
Control Activity ◽  
Transmembrane Segments ◽  
General Stress ◽  
Intermediate Metabolites ◽  
Primary Determinant ◽  
High Concentrations ◽  
Major Facilitator

2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113. The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites. The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments. PhlE has been suggested to function in PHL export. Here the sequencing of the phlE gene from P. fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported. It is shown that F113-D3 produced less PHL than F113. The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL. The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance. Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses. Osmotic stress induced rapid degradation of free PHL by the bacteria. Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells.

scholarly journals Structural and Functional Study of the Phenicol-Specific Efflux Pump FloR Belonging to the Major Facilitator Superfamily

2005 ◽  
Vol 49 (7) ◽  
pp. 2965-2971 ◽  
Author(s):  
Martine Braibant ◽  
Jacqueline Chevalier ◽  
Elisabeth Chaslus-Dancla ◽  
Jean-Marie Pagès ◽  
Axel Cloeckaert
Keyword(s):  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Site Directed Mutagenesis ◽  
Functional Study ◽  
Efflux Transporters ◽  
Chloramphenicol Resistance ◽  
Active Efflux ◽  
Transmembrane Segments ◽  
Efflux Mechanism ◽  
Major Facilitator

ABSTRACT The florfenicol-chloramphenicol resistance gene floR from Salmonella enterica was previously identified and postulated to belong to the major facilitator (MF) superfamily of drug exporters. Here, we confirmed a computer-predicted transmembrane topological model of FloR, using the phoA gene fusion method, and classified this protein in the DHA12 family (containing 12 transmembrane domains) of MF efflux transporters. We also showed that FloR is a transporter specific for structurally associated phenicol drugs (chloramphenicol, florfenicol, thiamphenicol) which utilizes the proton motive force to energize an active efflux mechanism. By site-directed mutagenesis of specific charged residues belonging to putative transmembrane segments (TMS), two residues essential for active efflux function, D23 in TMS1 and R109 in TMS4, were identified. Of these, the acidic residue D23 seems to participate directly in the affinity pocket involved in phenicol derivative recognition. A third residue, E283 in TMS9, seems to be necessary for correct membrane folding of the transporter.

scholarly journals Identification of anN-Acetylglucosamine Transporter That Mediates Hyphal Induction inCandida albicans

2007 ◽  
Vol 18 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Francisco J. Alvarez ◽  
James B. Konopka
Keyword(s):  
Plasma Membrane ◽  
Hyphal Growth ◽  
Nutrient Sensing ◽  
Major Facilitator Superfamily ◽  
Cellular Processes ◽  
Macrophage Phagocytosis ◽  
Wide Range ◽  
High Concentrations ◽  
Hyphal Formation ◽  
Major Facilitator

The sugar N-acetylglucosamine (GlcNAc) plays an important role in nutrient sensing and cellular regulation in a wide range of organisms from bacteria to humans. In the fungal pathogen Candida albicans, GlcNAc induces a morphological transition from budding to hyphal growth. Proteomic comparison of plasma membrane proteins from buds and from hyphae induced by GlcNAc identified a novel hyphal protein (Ngt1) with similarity to the major facilitator superfamily of transporters. An Ngt1-GFP fusion was detected in the plasma membrane after induction with GlcNAc, but not other related sugars. Ngt1-GFP was also induced by macrophage phagocytosis, suggesting a role for the GlcNAc response in signaling entry into phagolysosomes. NGT1 is needed for efficient GlcNAc uptake and for the ability to induce hyphae at low GlcNAc concentrations. High concentrations of GlcNAc could bypass the need for NGT1 to induce hyphae, indicating that elevated intracellular levels of GlcNAc induce hyphal formation. Expression of NGT1 in Saccharomyces cerevisiae promoted GlcNAc uptake, indicating that Ngt1 acts directly as a GlcNAc transporter. Transport mediated by Ngt1 was specific, as other sugars could not compete for the uptake of GlcNAc. Thus, Ngt1 represents the first eukaryotic GlcNAc transporter to be discovered. The presence of NGT1 homologues in the genome sequences of a wide range of eukaryotes from yeast to mammals suggests that they may also function in the cellular processes regulated by GlcNAc, including those that underlie important diseases such as cancer and diabetes.

scholarly journals Mdt(A), a New Efflux Protein Conferring Multiple Antibiotic Resistance in Lactococcus lactis andEscherichia coli

2001 ◽  
Vol 45 (4) ◽  
pp. 1109-1114 ◽  
Author(s):  
Vincent Perreten ◽  
Franziska V. Schwarz ◽  
Michael Teuber ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Lactococcus Lactis ◽  
Major Facilitator Superfamily ◽  
Multiple Drug ◽  
Drug Transporter ◽  
Multiple Antibiotic Resistance ◽  
Atp Binding Site ◽  
E Coli ◽  
Transmembrane Segments ◽  
Major Facilitator

ABSTRACT The mdt(A) gene, previously designatedmef214, from Lactococcus lactis subsp.lactis plasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactis and Escherichia coli, but not in Enterococcus faecalis or inStaphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactisand in E. coli.

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 Transcriptional Activation of ydeA, Which Encodes a Member of the Major Facilitator Superfamily, Interferes with Arabinose Accumulation and Induction of the Escherichia coli Arabinose PBADPromoter

1999 ◽  
Vol 181 (7) ◽  
pp. 2185-2191 ◽  
Author(s):  
Sandrine Bost ◽  
Filo Silva ◽  
Dominique Belin
Keyword(s):  
Transcriptional Activation ◽  
Growth Conditions ◽  
Initiation Site ◽  
Large Deletion ◽  
Major Facilitator Superfamily ◽  
Detectable Effect ◽  
Multicopy Plasmid ◽  
Transmembrane Segments ◽  
Negative Effect ◽  
Major Facilitator

ABSTRACT Induction of genes expressed from the arabinose PBADpromoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to theydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient −35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). TheydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of ydeA has no detectable effect on cell growth. Thus, ydeA appears to be nonessential under usual laboratory growth conditions. The ydeA gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect ofydeA transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.

scholarly journals Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter

2003 ◽  
Vol 372 (1) ◽  
pp. 247-252 ◽  
Author(s):  
Toshiko KASAHARA ◽  
Michihiro KASAHARA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Amino Acid Identity ◽  
Major Facilitator Superfamily ◽  
Transport Activity ◽  
Transmembrane Segments ◽  
High Affinity ◽  
Yeast Transformants ◽  
Major Facilitator

Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily. Hxt1 shares ≈ 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports d-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The Km and Vmax values for d-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2.

scholarly journals A Major Facilitator Superfamily Transporter Plays a Dual Role in Polar Auxin Transport and Drought Stress Tolerance in Arabidopsis

2013 ◽  
Vol 25 (3) ◽  
pp. 901-926 ◽  
Author(s):  
Estelle Remy ◽  
Tânia R. Cabrito ◽  
Pawel Baster ◽  
Rita A. Batista ◽  
Miguel C. Teixeira ◽  
...  
Keyword(s):  
Drought Stress ◽  
Stress Tolerance ◽  
Auxin Transport ◽  
Polar Auxin Transport ◽  
Dual Role ◽  
Major Facilitator Superfamily ◽  
Drought Stress Tolerance ◽  
Major Facilitator Superfamily Transporter ◽  
Major Facilitator

Multidrug resistant phenotype of Bacillus subtilis spontaneous mutants isolated in the presence of puromycin and lincomycin

2003 ◽  
Vol 49 (2) ◽  
pp. 71-77 ◽  
Author(s):  
Makiko Murata ◽  
Shinichi Ohno ◽  
Miyuki Kumano ◽  
Kunio Yamane ◽  
Reiko Ohki
Keyword(s):  
Bacillus Subtilis ◽  
Multidrug Resistant ◽  
Major Facilitator Superfamily ◽  
Northern Hybridization ◽  
Bacillus Subtilis 168 ◽  
Resistant Phenotype ◽  
High Concentrations ◽  
Major Facilitator ◽  
Spontaneous Mutants ◽  
Lincomycin Resistance

Spontaneous mutants were isolated by growing Bacillus subtilis 168 in the presence of high concentrations of puromycin and lincomycin. These mutants showed increased resistance to several drugs other than these two drugs. The lmrAB genes, which encode a transcriptional repressor and a drug efflux protein of the major facilitator superfamily, were involved in this phenotype. Northern hybridization analysis showed that the expression of lmrAB gene increased more than 30-fold. The following two types of mutations were found to be responsible for the multidrug resistant phenotype: (i) a nucleotide replacement in the region between the promoter and initiation codon of lmrA and (ii) nucleotide replacements that resulted in amino acid replacements in the LmrA protein. The results indicate that LmrB is a multidrug resistant protein and that LmrA is a repressor, which autogenously represses the transcription of the lmrAB operon.Key words: multidrug resistance (MDR), major facilitator superfamily (MFS), lincomycin resistance, lmrAB genes.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

Sign in / Sign up

Export Citation Format

Share Document