Subpopulation-Specific Metabolic Pathway Usage in Mixed Cultures as Revealed by Reporter Protein-Based 13 C Analysis

Abstract

ABSTRACT Most large-scale biological processes, like global element cycling or decomposition of organic matter, are mediated by microbial consortia. Commonly, the different species in such consortia exhibit mutual metabolic dependencies that include the exchange of nutrients. Despite the global importance, surprisingly little is known about the metabolic interplay between species in particular subpopulations. To gain insight into the intracellular fluxes of subpopulations and their interplay within such mixed cultures, we developed here a 13 C flux analysis approach based on affinity purification of the recombinant fusion glutathione S -transferase (GST) and green fluorescent protein (GFP) as a reporter protein. Instead of detecting the 13 C labeling patterns in the typically used amino acids from the total cellular protein, our method detects these 13 C patterns in amino acids from the reporter protein that has been expressed in only one species of the consortium. As a proof of principle, we validated our approach by mixed-culture experiments of an Escherichia coli wild type with two metabolic mutants. The reporter method quantitatively resolved the expected mutant-specific metabolic phenotypes down to subpopulation fractions of about 1%.