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Fitness-enhancing adaptations of protein expression and its regulation are an important aspect of bacterial evolution. A key question is whether evolution has led to optimal protein expression that maximizes immediate growth rate (short-term fitness) in a robust manner (consistently across diverse conditions). Alternatively, they could display suboptimal short-term fitness, because they cannot do better or because they instead strive for long-term fitness maximization by, for instance, preparing for future conditions. To address this question, we focus on the ATP-producing enzyme F1F0 H+-ATPase, which is an abundant enzyme and ubiquitously expressed across conditions. Its expression is highly regulated and dependent on growth rate and nutrient conditions. For instance, during growth on sugars, when metabolism is overflowing acetate, glycolysis supplies most ATP, while H+-ATPase is the main source of ATP synthesis during growth on acetate. We tested the optimality of H+-ATPase expression in Escherichia coli across different nutrient conditions. In all tested conditions, wild-type E. coli expresses its H+-ATPase remarkably close (within a few per cent) to optimal concentrations that maximize immediate growth rate. This work indicates that bacteria can indeed achieve robust optimal protein expression for immediate growth-rate maximization.

Overflow metabolism is ubiquitous in nature, and it is often considered inefficient because it leads to a relatively low biomass yield per consumed carbon. This metabolic strategy has been described as advantageous because it supports high growth rates during nutrient competition. Here, we experimentally evolved bacteria without nutrient competition by repeatedly growing and mixing millions of parallel batch cultures of Escherichia coli. Each culture originated from a water-in-oil emulsion droplet seeded with a single cell. Unexpectedly we found that overflow metabolism (acetate production) did not change. Instead, the numerical cell yield during the consumption of the accumulated acetate increased as a consequence of a reduction in cell size. Our experiments and a mathematical model show that fast growth and overflow metabolism, followed by the consumption of the overflow metabolite, can lead to a higher numerical cell yield and therefore a higher fitness compared with full respiration of the substrate. This provides an evolutionary scenario where overflow metabolism can be favorable even in the absence of nutrient competition.