Identification of a c-Type Cytochrome Specific for Manganese Dioxide (MnO2) Reduction in Anaeromyxobacter dehalogenans Strain 2CP-C

Abstract:

Anaeromyxobacter dehalogenans is a metabolically versatile Deltaproteobacterium and conserves energy from the reduction of various electron acceptors, including insoluble MnO₂ and ferric oxides/oxyhydroxides (FeOOH). The goal of this study was to identify c-type cytochromes involved in electron transfer to MnO₂. The characterization of deletion mutants has revealed a number of c-type cytochromes involved in electron transfer to solid metal oxides in Shewanella spp. and Geobacter spp; however, a genetic system for Anaeromyxobacter is not available. The A. dehalogenans str. 2CP-C genome encodes 68 putative c-type cytochromes, which all lack functional assignments. To identify c-type cytochromes involved in electron transfer to solid MnO₂, protein expression profiles of A. dehalogenans str. 2CP-C cells grown with acetate as electron donor and MnO₂, ferric citrate, FeOOH, nitrate or fumarate as electron acceptors were compared. Whole cell proteomes were analyzed after trypsin proteolysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Distinct c-type cytochrome expression patterns were observed with cells grown with different electron acceptors. A. dehalogenans str. 2CP-C grown with MnO₂ expressed 25 out of the 68 c-type cytochromes encoded on the genome. The c-type cytochrome Adeh_1278 was only expressed in strain 2CP-C grown with MnO₂. Reverse transcription PCR confirmed that the Adeh_1278 gene was transcribed in MnO₂-grown cells but not in cells grown with other terminal electron acceptors. The expression of the Adeh_1278 gene correlated with Mn(IV) reduction activity. Adeh_1278 has three heme binding motifs and is predicted to be located in the periplasm. The identification of Adeh_1278 as a protein uniquely expressed when MnO₂ serves as electron acceptor suggests its utility as a biomarker for MnO₂ reduction. This example demonstrates the value of the LC-MS/MS approach for identifying specific proteins of interest and making functional assignments to proteins, including c-type cytochromes that have not been characterized. The distinctive expression of c-type cytochromes in response to growth with different terminal electron acceptors offers opportunities for functional (i.e., activity) in situ monitoring using metaproteomics or transcript-targeted approaches.