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Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction

¹ Department of Microbiology, 203N Morrill Science Center IVN, University of Massachusetts at Amherst, Amherst, MA 01003, USA
² Ciba Specialty Chemicals Ltd, Klybeckstr. 141, CH-4002 Basel, Switzerland
³ The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850, USA
⁴ Department of Mathematics and Statistics, Lederle Graduate Research Tower, Box 34515, University of Massachusetts at Amherst, Amherst, MA 01003, USA

Correspondence
Derek R. Lovley
dlovley{at}microbio.umass.edu

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 Geobacter sulfurreducens, 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 in vivo is unlikely and suggest that deleting SfrAB may interfere with growth via acetate oxidation by interfering with NADP regeneration.

Three supplementary figures, and supplementary tables of the primers used in QRT-PCR and genes that are up- or downregulated in the acetate-adapted SfrAB-null strain during growth in chemostats in acetate : fumarate medium are available with the online version of this paper, together with an overview and additional discussion of the microarray analysis.