Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures

It is still a challenge to link specific metabolic activities to certain species in a microbial community because of methodological limitations. We developed a method to analyze the specific metabolic activity of a single bacterial species within a consortium making use of [(13)C(7)]-toluene for metabolic labelling of proteins. Labelled proteins were subsequently analyzed by 2D gel electrophoresis (2-DE) and mass spectrometry (MS) to characterize their identity as well as their (13)C content as an indicator for function and activity of the host organism. To establish this method, we analyzed the metabolic incorporation of (13)C carbon atoms into proteins of Aromatoleum aromaticum strain EbN1. This strain is capable of metabolizing toluene under nitrate-reducing conditions and was grown in either pure culture or in a mixed consortium with a gluconate-consuming enrichment culture. First, strain EbN1 was grown with non-labelled toluene or labelled [(13)C(7)]-toluene as carbon sources, respectively, and their proteins were subjected to 2-DE. In total, 60 unique proteins were identified by MALDI-MS/MS. From 38 proteins, the levels of (13)C incorporation were determined as 92.3+/-0.8%. Subsequently, we mixed strain EbN1 and the enrichment culture UFZ-1, which does not grow on toluene but on gluconate, and added non-labelled toluene, [(13)C(7)]-toluene and/or non-labelled gluconate as carbon sources. The isotope labelling of proteins was analyzed after 2-DE by MS as a quantitative indicator for metabolic transformation of isotopic-labelled toluene by the active species of the consortium. Incorporation of (13)C was exclusively found in proteins from strain EbN1 at a content of 82.6+/-2.3%, as an average calculated from 19 proteins, demonstrating the suitability of the method used to identify metabolic active species with specific properties within a mixed culture.