Metabolism in dense microbial colonies: 13C metabolic flux analysis of E. coli grown on agar identifies two distinct cell populations with acetate crossfeeding

In this study, we have investigated for the first time the metabolism of E. coli grown on agar using C-13 metabolic flux analysis (C-13-MFA). To date, all C-13-MFA studies on microbes have been performed with cells grown in liquid culture. Here, we extend the scope of C-13-MFA to biological systems where cells are grown in dense microbial colonies. First, we identified new optimal C-13 tracers to quantify fluxes in systems where the acetate yield cannot be easily measured. We determined that three parallel labeling experiments with the tracers [1,2-C-13] glucose, [1,6-C-13] glucose, and [4,5,6-C-13] glucose permit precise estimation of not only intracellular fluxes, but also of the amount of acetate produced from glucose. Parallel labeling experiments were then performed with wild-type E. coli and E. coli.ackA grown in liquid culture and on agar plates. Initial attempts to fit the labeling data from wild-type E. coli grown on agar did not produce a statistically acceptable fit. To resolve this issue, we employed the recently developed co-culture C-13-MFA approach, where two E. coli subpopulations were defined in the model that engaged in metabolite cross-feeding. The flux results identified two distinct E. coli cell populations, a dominant cell population (92% of cells) that metabolized glucose via conventional metabolic pathways and secreted a large amount of acetate (similar to 40% of maximum theoretical yield), and a second smaller cell population (8% of cells) that consumed the secreted acetate without any glucose influx. These experimental results are in good agreement with recent theoretical simulations. Importantly, this study provides a solid foundation for future investigations of a wide range of problems involving microbial biofilms that are of great interest in biotechnology, ecology and medicine, where metabolite cross-feeding between cell populations is a core feature of the communities.