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1.
Genetics ; 169(1): 107-22, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15489514

ABSTRACT

We screened a collection of 4847 haploid knockout strains (EUROSCARF collection) of Saccharomyces cerevisiae for iron uptake from the siderophore ferrioxamine B (FOB). A large number of mutants showed altered uptake activities, and a few turned yellow when grown on agar plates with added FOB, indicating increased intracellular accumulation of undissociated siderophores. A subset consisting of 197 knockouts with altered uptake was examined further for regulated activities that mediate cellular uptake of iron from other siderophores or from iron salts. Hierarchical clustering analysis grouped the data according to iron sources and according to mutant categories. In the first analysis, siderophores grouped together with the exception of enterobactin, which grouped with iron salts, suggesting a reductive pathway of iron uptake for this siderophore. Mutant groupings included three categories: (i) high-FOB uptake, high reductase, low-ferrous transport; (ii) isolated high- or low-FOB transport; and (iii) induction of all activities. Mutants with statistically altered uptake activities included genes encoding proteins with predominant localization in the secretory pathway, nucleus, and mitochondria. Measurements of different iron-uptake activities in the yeast knockout collection make possible distinctions between genes with general effects on iron metabolism and those with pathway-specific effects.


Subject(s)
Deferoxamine/metabolism , Ferric Compounds/metabolism , Genome, Fungal , Iron/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Siderophores/metabolism , Biological Transport , Cluster Analysis , Deferoxamine/pharmacokinetics , Enterobactin/metabolism , Enterobactin/pharmacokinetics , FMN Reductase/metabolism , Ferric Compounds/pharmacokinetics , Iron/pharmacokinetics , Iron Chelating Agents/metabolism , Iron Chelating Agents/pharmacokinetics , Mutation/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Siderophores/pharmacokinetics
2.
J Biol Chem ; 276(13): 10218-23, 2001 Mar 30.
Article in English | MEDLINE | ID: mdl-11120744

ABSTRACT

Saccharomyces cerevisiae takes up siderophore-bound iron through two distinct systems, one that requires siderophore transporters of the ARN family and one that requires the high affinity ferrous iron transporter on the plasma membrane. Uptake through the plasma membrane ferrous iron transporter requires that the iron first must dissociate from the siderophore and undergo reduction to the ferrous form. FRE1 and FRE2 encode cell surface metalloreductases that are required for reduction and uptake of free ferric iron. The yeast genome contains five additional FRE1 and FRE2 homologues, four of which are regulated by iron and the major iron-dependent transcription factor, Aft1p, but whose function remains unknown. Fre3p was required for the reduction and uptake of ferrioxamine B-iron and for growth on ferrioxamine B, ferrichrome, triacetylfusarinine C, and rhodotorulic acid in the absence of Fre1p and Fre2p. By indirect immunofluorescence, Fre3p was expressed on the plasma membrane in a pattern similar to that of Fet3p, a component of the high affinity ferrous transporter. Enterobactin, a catecholate siderophore, was not a substrate for Fre3p, and reductive uptake required either Fre1p or Fre2p. Fre4p could facilitate utilization of rhodotorulic acid-iron when the siderophore was present in higher concentrations. We propose that Fre3p and Fre4p are siderophore-iron reductases and that the apparent redundancy of the FRE genes confers the capacity to utilize iron from a variety of siderophore sources.


Subject(s)
Cell Membrane/enzymology , FMN Reductase , Iron/pharmacokinetics , Membrane Transport Proteins , Oxidoreductases/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Siderophores/pharmacokinetics , Carrier Proteins/metabolism , Deferoxamine/metabolism , Dose-Response Relationship, Drug , Enterobactin/metabolism , Enterobactin/pharmacokinetics , Ferric Compounds/metabolism , Ferrichrome/metabolism , Fluorescent Antibody Technique, Indirect , Fungal Proteins/metabolism , Iron/metabolism , Microscopy, Fluorescence , NADH, NADPH Oxidoreductases/genetics , Oxidoreductases/metabolism , Piperazines/metabolism , Plasmids/metabolism , Siderophores/metabolism , Transcription Factors/metabolism
3.
Microbiology (Reading) ; 141 ( Pt 12): 3181-91, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8574410

ABSTRACT

Siderophore-mediated iron uptake systems play a central role in the pathogenesis of infection for many bacterial pathogens. Campylobacter species are not thought to produce siderophores, yet they are able to utilize both ferrichrome and enterochelin as sources of iron. Part of an operon named ceuBCDE, encoding components of a periplasmic binding-protein-dependent transport (PBT) system for the uptake of a ferric siderophore from Campylobacter coli, was cloned directly into Escherichia coli using a plasmid rescue technique. Phenotypic and genetic analyses of this system showed it to comprise two hydrophobic integral membrane proteins, CeuB (35.5 kDa) and CeuC (34.8 kDa), which may form the cytoplasmic membrane permease, an ATP-binding protein, CeuD (28.8 kDa), and a periplasmic substrate-binding protein, CeuE (34.5 kDa). In vivo labelling studies using [3H]palmitate demonstrated that CeuE, the periplasmic binding protein, is expressed as a lipoprotein in C. coli, which is unusual for a Gram-negative PBT system. Mutants of C. coli, defective in components of the transport mechanism, were severely impaired in the ability to utilize enterochelin as an iron source suggesting that this siderophore is a substrate for the transport system. This is the first molecular characterization of a PBT system in Campylobacter species.


Subject(s)
Bacterial Outer Membrane Proteins , Bacterial Proteins/metabolism , Campylobacter coli/metabolism , Carrier Proteins/metabolism , Enterobactin/pharmacokinetics , Receptors, Cell Surface , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Biological Transport, Active , Campylobacter coli/genetics , Carrier Proteins/genetics , Cloning, Molecular , DNA Primers/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Genes, Bacterial , Lipoproteins/genetics , Lipoproteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Operon , Plasmids/genetics , Restriction Mapping
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