1887

Abstract

A soluble ferric reductase, SfrAB, which catalysed the NADPH-dependent reduction of chelated Fe(III), was previously purified from the dissimilatory Fe(III)-reducing micro-organism suggesting that reduction of chelated forms of Fe(III) might be cytoplasmic. However, metabolically active spheroplast suspensions could not catalyse acetate-dependent Fe(III) citrate reduction, indicating that periplasmic and/or outer-membrane components were required for Fe(III) citrate reduction. Furthermore, phenotypic analysis of an SfrAB knockout mutant suggested that SfrAB was involved in acetate metabolism rather than respiration-linked Fe(III) reduction. The mutant could not grow via the reduction of either Fe(III) citrate or fumarate when acetate was the electron donor but could grow with either acceptor if either hydrogen or formate served as the electron donor. Following prolonged incubation in acetate : fumarate medium in the absence of hydrogen and formate, an ‘acetate-adapted’ SfrAB-null strain was isolated that was capable of growth on acetate : fumarate medium but not acetate : Fe(III) citrate medium. Comparison of gene expression in this strain with that of the wild-type revealed upregulation of a potential NADPH-dependent ferredoxin oxidoreductase as well as genes involved in energy generation and amino acid uptake, suggesting that NADPH homeostasis and the tricarboxylic acid (TCA) cycle were perturbed in the ‘acetate-adapted’ SfrAB-null strain. Membrane and soluble fractions prepared from the ‘acetate-adapted’ strain were depleted of NADPH-dependent Fe(III), viologen and quinone reductase activities. These results indicate that cytoplasmic, respiration-linked reduction of Fe(III) by SfrAB is unlikely and suggest that deleting SfrAB may interfere with growth via acetate oxidation by interfering with NADP regeneration.

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QRT-PCR primers [ PDF] (14 kb) Genes that are upregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (22 kb) Genes that are downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (40 kb) Growth of the wild-type srain and an independently isolated, acetate-adapted SfrAB-null strain in (A) acetate:fumarate (15 mM:20mM) medium and (B) acetate:Fe(III) (20 mM:55 mM) medium. Inocula (3%) were exponential-phase acetate:fumarate cultures. Data are means and standard deviations of triplicate cultures. PowerPoint file(68 kb) Preliminary biochemical characterization of an independently isolated, acetate-adapted SfrAB-null strain. (A) Determination of NADPH-dependent Fe(III)-NTA, AQDS, and benzyl viologen reductase activities in membrane and soluble fractions prepared from the wild-type and SfrAB-null strains. (B) Distribution of the cytoplasmic enzyme, oxoglutarate/ferredoxin oxidoreductase, and the various reductase activities in the membrane and soluble fractions of the wild-type strain. PowerPoint file(50 kb) Functional characterization of genes that were differentially expressed in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium and had a fold change in expression of >1.5 or <-1.5 relative to wild type. Functional categories were created and assigned by the authors based on analysis of protein sequences. The category Unknown includes both hypothetical proteins and conserved proteins of unknown function. PowerPoint file(63 kb) [ PDF] (492 kb)

PDF

QRT-PCR primers [ PDF] (14 kb) Genes that are upregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (22 kb) Genes that are downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (40 kb) Growth of the wild-type srain and an independently isolated, acetate-adapted SfrAB-null strain in (A) acetate:fumarate (15 mM:20mM) medium and (B) acetate:Fe(III) (20 mM:55 mM) medium. Inocula (3%) were exponential-phase acetate:fumarate cultures. Data are means and standard deviations of triplicate cultures. PowerPoint file(68 kb) Preliminary biochemical characterization of an independently isolated, acetate-adapted SfrAB-null strain. (A) Determination of NADPH-dependent Fe(III)-NTA, AQDS, and benzyl viologen reductase activities in membrane and soluble fractions prepared from the wild-type and SfrAB-null strains. (B) Distribution of the cytoplasmic enzyme, oxoglutarate/ferredoxin oxidoreductase, and the various reductase activities in the membrane and soluble fractions of the wild-type strain. PowerPoint file(50 kb) Functional characterization of genes that were differentially expressed in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium and had a fold change in expression of >1.5 or <-1.5 relative to wild type. Functional categories were created and assigned by the authors based on analysis of protein sequences. The category Unknown includes both hypothetical proteins and conserved proteins of unknown function. PowerPoint file(63 kb) [ PDF] (492 kb)

PDF

QRT-PCR primers [ PDF] (14 kb) Genes that are upregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (22 kb) Genes that are downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (40 kb) Growth of the wild-type srain and an independently isolated, acetate-adapted SfrAB-null strain in (A) acetate:fumarate (15 mM:20mM) medium and (B) acetate:Fe(III) (20 mM:55 mM) medium. Inocula (3%) were exponential-phase acetate:fumarate cultures. Data are means and standard deviations of triplicate cultures. PowerPoint file(68 kb) Preliminary biochemical characterization of an independently isolated, acetate-adapted SfrAB-null strain. (A) Determination of NADPH-dependent Fe(III)-NTA, AQDS, and benzyl viologen reductase activities in membrane and soluble fractions prepared from the wild-type and SfrAB-null strains. (B) Distribution of the cytoplasmic enzyme, oxoglutarate/ferredoxin oxidoreductase, and the various reductase activities in the membrane and soluble fractions of the wild-type strain. PowerPoint file(50 kb) Functional characterization of genes that were differentially expressed in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium and had a fold change in expression of >1.5 or <-1.5 relative to wild type. Functional categories were created and assigned by the authors based on analysis of protein sequences. The category Unknown includes both hypothetical proteins and conserved proteins of unknown function. PowerPoint file(63 kb) [ PDF] (492 kb)

PDF

QRT-PCR primers [ PDF] (14 kb) Genes that are upregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (22 kb) Genes that are downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (40 kb) Growth of the wild-type srain and an independently isolated, acetate-adapted SfrAB-null strain in (A) acetate:fumarate (15 mM:20mM) medium and (B) acetate:Fe(III) (20 mM:55 mM) medium. Inocula (3%) were exponential-phase acetate:fumarate cultures. Data are means and standard deviations of triplicate cultures. PowerPoint file(68 kb) Preliminary biochemical characterization of an independently isolated, acetate-adapted SfrAB-null strain. (A) Determination of NADPH-dependent Fe(III)-NTA, AQDS, and benzyl viologen reductase activities in membrane and soluble fractions prepared from the wild-type and SfrAB-null strains. (B) Distribution of the cytoplasmic enzyme, oxoglutarate/ferredoxin oxidoreductase, and the various reductase activities in the membrane and soluble fractions of the wild-type strain. PowerPoint file(50 kb) Functional characterization of genes that were differentially expressed in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium and had a fold change in expression of >1.5 or <-1.5 relative to wild type. Functional categories were created and assigned by the authors based on analysis of protein sequences. The category Unknown includes both hypothetical proteins and conserved proteins of unknown function. PowerPoint file(63 kb) [ PDF] (492 kb)

POWERPOINT

QRT-PCR primers [ PDF] (14 kb) Genes that are upregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (22 kb) Genes that are downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (40 kb) Growth of the wild-type srain and an independently isolated, acetate-adapted SfrAB-null strain in (A) acetate:fumarate (15 mM:20mM) medium and (B) acetate:Fe(III) (20 mM:55 mM) medium. Inocula (3%) were exponential-phase acetate:fumarate cultures. Data are means and standard deviations of triplicate cultures. PowerPoint file(68 kb) Preliminary biochemical characterization of an independently isolated, acetate-adapted SfrAB-null strain. (A) Determination of NADPH-dependent Fe(III)-NTA, AQDS, and benzyl viologen reductase activities in membrane and soluble fractions prepared from the wild-type and SfrAB-null strains. (B) Distribution of the cytoplasmic enzyme, oxoglutarate/ferredoxin oxidoreductase, and the various reductase activities in the membrane and soluble fractions of the wild-type strain. PowerPoint file(50 kb) Functional characterization of genes that were differentially expressed in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium and had a fold change in expression of >1.5 or <-1.5 relative to wild type. Functional categories were created and assigned by the authors based on analysis of protein sequences. The category Unknown includes both hypothetical proteins and conserved proteins of unknown function. PowerPoint file(63 kb) [ PDF] (492 kb)

POWERPOINT

QRT-PCR primers [ PDF] (14 kb) Genes that are upregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (22 kb) Genes that are downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (40 kb) Growth of the wild-type srain and an independently isolated, acetate-adapted SfrAB-null strain in (A) acetate:fumarate (15 mM:20mM) medium and (B) acetate:Fe(III) (20 mM:55 mM) medium. Inocula (3%) were exponential-phase acetate:fumarate cultures. Data are means and standard deviations of triplicate cultures. PowerPoint file(68 kb) Preliminary biochemical characterization of an independently isolated, acetate-adapted SfrAB-null strain. (A) Determination of NADPH-dependent Fe(III)-NTA, AQDS, and benzyl viologen reductase activities in membrane and soluble fractions prepared from the wild-type and SfrAB-null strains. (B) Distribution of the cytoplasmic enzyme, oxoglutarate/ferredoxin oxidoreductase, and the various reductase activities in the membrane and soluble fractions of the wild-type strain. PowerPoint file(50 kb) Functional characterization of genes that were differentially expressed in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium and had a fold change in expression of >1.5 or <-1.5 relative to wild type. Functional categories were created and assigned by the authors based on analysis of protein sequences. The category Unknown includes both hypothetical proteins and conserved proteins of unknown function. PowerPoint file(63 kb) [ PDF] (492 kb)

POWERPOINT

QRT-PCR primers [ PDF] (14 kb) Genes that are upregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (22 kb) Genes that are downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium [ PDF] (40 kb) Growth of the wild-type srain and an independently isolated, acetate-adapted SfrAB-null strain in (A) acetate:fumarate (15 mM:20mM) medium and (B) acetate:Fe(III) (20 mM:55 mM) medium. Inocula (3%) were exponential-phase acetate:fumarate cultures. Data are means and standard deviations of triplicate cultures. PowerPoint file(68 kb) Preliminary biochemical characterization of an independently isolated, acetate-adapted SfrAB-null strain. (A) Determination of NADPH-dependent Fe(III)-NTA, AQDS, and benzyl viologen reductase activities in membrane and soluble fractions prepared from the wild-type and SfrAB-null strains. (B) Distribution of the cytoplasmic enzyme, oxoglutarate/ferredoxin oxidoreductase, and the various reductase activities in the membrane and soluble fractions of the wild-type strain. PowerPoint file(50 kb) Functional characterization of genes that were differentially expressed in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate:fumarate medium and had a fold change in expression of >1.5 or <-1.5 relative to wild type. Functional categories were created and assigned by the authors based on analysis of protein sequences. The category Unknown includes both hypothetical proteins and conserved proteins of unknown function. PowerPoint file(63 kb) [ PDF] (492 kb)

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