Abstract
Desulfotomaculum reducens strain MI-1 is a Gram-positive, sulfate-reducing bacterium also capable of reducing Fe(III). Metal reduction in Gram-positive bacteria is poorly understood. Here, we investigated Fe(III) reduction with lactate, a non-fermentable substrate, as the electron donor. Lactate consumption is concomitant to Fe(III) reduction, but does not support significant growth, suggesting that little energy can be conserved from this process and that it may occur fortuitously. D. reducens can reduce both soluble [Fe(III)-citrate] and insoluble (hydrous ferric oxide, HFO) Fe(III). Because physically inaccessible HFO was not reduced, we concluded that reduction requires direct contact under these experimental conditions. This implies the presence of a surface exposed reductase capable of transferring electrons from the cell to the extracellular electron acceptor. With the goal of characterizing the role of surface proteins in D. reducens and of identifying candidate Fe(III) reductases, we carried out an investigation of the surface proteome (surfaceome) of D. reducens. Cell surface exposed proteins were extracted by trypsin cell shaving or by lysozyme treatment, and analyzed by liquid chromatography-tandem mass spectrometry. This investigation revealed that the surfaceome fulfills many functions, including solute transport, protein export, maturation and hydrolysis, peptidoglycan synthesis and modification, and chemotaxis. Furthermore, a few redox-active proteins were identified. Among these, three are putatively involved in Fe(III) reduction, i.e., a membrane-bound hydrogenase 4Fe-4S cluster subunit (Dred_0462), a heterodisulfide reductase subunit A (Dred_0143) and a protein annotated as alkyl hydroperoxide reductase but likely functioning as a thiol-disulfide oxidoreductase (Dred_1533).
Highlights
The cell structure of Gram-positive bacteria is characterized by a single membrane surrounded by a relatively thick cell wall (CW)
We found that neither method was completely successful in enriching for solely surface exposed proteins: a total of 599 proteins were extracted in the protoplast experiment, and 767 were initially identified by the MaxQuant software in the shaving experiment, but this dataset was reduced to 469 by a 1% false-discovery rate (FDR)
D. reducens is known to be capable of metal reduction (Tebo and Obraztsova, 1998)
Summary
The cell structure of Gram-positive bacteria is characterized by a single membrane surrounded by a relatively thick cell wall (CW). Other important roles for surface proteins include: adhesion to substrates and intercellular interaction for biofilm formation; environmental signal reception and activation of a response (e.g., chemotaxis, stress response); motility (flagella); intercellular genetic exchange (i.e., conjugation through pili); extracellular substrate binding for transport to the cytoplasm; CW digestion, either self-directed as in the case of autolysins, or aimed at cells of other species as a defense mechanism (Smith et al, 2000; Desvaux et al, 2006) Another role most likely played by surface proteins of Gram-positive bacteria is extracellular electron transfer. Carlson et al identified a surface-exposed multi-heme c-type cytochrome that is putatively involved in extracellular Fe(III) reduction in Thermincola potens JR (Carlson et al, 2012)
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