(163d) Thermodynamic Driving Force and Protein Cost Complement the Ccpa Regulation Governing the Onset of Staphylococcus Aureus Overflow Metabolism

Staphylococcus aureus, a severe human pathogen, distinctively exhibits overflow metabolism at low growth rates, which is a puzzling phenotype from the bioenergetics perspective. Therefore, this study employs a synergistic experimental and modeling approach to investigate this interesting trait. First, to revisit the known suppression of the TCA cycle by ccpA, a ΔccpA mutant of S. aureus was created. However, the observed acetate overflow in the ΔccpA mutant indicated the involvement of other regulatory mechanisms. To find these regulations, we applied an in-silico analysis that identified succinate dehydrogenase, mediated by menaquinone, as a key thermodynamic bottleneck in aerobic glucose catabolism, leading to obligatory acetate overflow. Additionally, a comparative protein cost analysis of the central carbon pathway highlighted the higher resource demand of the TCA cycle over the acetate overflow pathway. Moreover, our novel algorithm, 'InteGraM' when combined with the OptMDFpathway framework, indicated that membrane crowding limits electron transport chain activity. This limitation increases dependence on acetate overflow for ATP production, a trend consistent with the membrane-real estate hypothesis. The modeling result was again verified by ¹³C labeling experiments. Overall, this study demonstrates the importance of thermodynamic driving forces and resource allocation, in addition to ccpA regulation, as governing principles of S. aureus’s onset of overflow metabolism.