Aerobicity stimulon in Escherichia coli revealed using multi-scale computational systems biology of respiratory variants

Energy homeostasis facilitated by the interplay of substrate-level and oxidative phosphorylation is crucial for bacterial adaptation to diverse substrates and environments. To investigate how bioenergetic systems optimize under metabolically restrictive conditions, we evolved electron transport system (ETS) variants with distinct proton-pumping efficiencies on succinate and glycerol. These substrates impose unique metabolic constraints: succinate requires complete gluconeogenesis, while glycerol supports mixed glycolytic and gluconeogenic fluxes. Multi-scale computational analysis of the strains revealed (a) Growth optimization across carbon substrates for multiple ETS variants, (b) A conserved aerobicity stimulon comprising seven independently regulated gene groups that are co-regulated with increasing aerobic capacities, (c) Proteome reallocation linked to aerobicity, validated using genome-scale metabolism and expression modeling, and (d) Carbon source-specific compensatory mutations. These findings define the aerobicity stimulon and establish a unifying framework for understanding bacterial respiratory flexibility, demonstrating how transcriptional networks and metabolic systems integrate to achieve energy homeostasis and bioenergetic resilience.