Research Article: A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in Syntrophomonas wolfei

Date Published: February 26, 2013

Publisher: Public Library of Science

Author(s): Alexander Schmidt, Nicolai Müller, Bernhard Schink, David Schleheck, Ivo G. Boneca.


In syntrophic conversion of butyrate to methane and CO2, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO2 reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate.

Partial Text

Fermentation of butyrate to methane and CO2 is catalyzed by fatty acid-oxidizing bacteria in syntrophic cooperation with hydrogen-scavenging, methanogenic partner organisms, e.g., by Syntrophomonas wolfei in cooperation with Methanospirillum hungatei. Under these conditions, the butyrate-oxidizing bacteria can gain energy in the range of approximately −20 kJ per mol of butyrate oxidized [1], which is just sufficient to support microbial growth [2]. However, the biochemical mechanism of syntrophic butyrate oxidation by S. wolfei has not yet been resolved [3].

Resolving the mystery of how Syntrophomonas wolfei couples fermentation of butyrate to acetate with hydrogen/formate formation, which is energetically unfavourable, is a particularly difficult challenge [17]. A very essential step forward was the sequencing and thorough annotation of the genome of S. wolfei[12]. Another recent and important step was the purification and identification of specific enzymes of S. wolfei, e.g., an NADH:acceptor oxidoreductase activity and butyryl-CoA dehydrogenase activity [14]. In the present study, all proteins in S. wolfei that are highly expressed during syntrophic growth with butyrate were compared by protein gel electrophoresis with those expressed during pure culture growth with crotonate, and identified by peptide fingerprinting-mass spectrometry (PF-MS). The rationale for our gel-based proteomic approach was that all highly abundant proteins visible on gels can be considered to be important for some aspects of cellular function, especially in an organism such as S. wolfei that grows under such a difficult energetic condition and therefore has to economize its energy consumption, e.g., in protein synthesis. Furthermore, our gel-based proteomic approach allowed to evaluate not only the relative abundance of proteins (by their band intensities), but also the PF-MS identifications obtained when comparing the observed molecular masses of the proteins (and their isoelectric points in case of 2D-gels) with the predicted molecular masses (and pI) derived of the respective attributed gene sequences. The results obtained (Figs. 1AB, 2AB, S1, S2 and Tables 1 and 2) in combination with results from Blue-Native PAGE and activity staining (Figs. 3, S3, S4, S5, S6, and S7 and Table S1 and S2) and enzyme measurements in intact cells and cell extracts (Tables 3 and 4) allow to derive a first evidence-based concept of the electron flow and energy economy for this unusual type of metabolism operating close to the minimum energy yield for microbial growth (Fig. 4).