New experimental results on bacterial growth inspire a novel top-down approach to study cell metabolism, combining mass balance and proteomic constraints to extend and complement Flux Balance Analysis. We introduce here Constrained Allocation Flux Balance Analysis, CAFBA, in which the biosynthetic costs associated to growth are accounted for in an effective way through a single additional genome-wide constraint. Its roots lie in the experimentally observed pattern of proteome allocation for metabolic functions, allowing to bridge regulation and metabolism in a transparent way under the principle of growth-rate maximization. We provide a simple method to solve CAFBA efficiently and propose an “ensemble averaging” procedure to account for unknown protein costs. Applying this approach to modeling E. coli metabolism, we find that, as the growth rate increases, CAFBA solutions cross over from respiratory, growth-yield maximizing states (preferred at slow growth) to fermentative states with carbon overflow (preferred at fast growth). In addition, CAFBA allows for quantitatively accurate predictions on the rate of acetate excretion and growth yield based on only 3 parameters determined by empirical growth laws. The intracellular protein levels of exponentially growing bacteria are known to vary strongly with growth conditions, as described by quantitative “growth laws”. This work introduces a computational genome-scale framework (Constrained Allocation Flux Balance Analysis, CAFBA) which incorporates growth laws into canonical Flux Balance Analysis. Upon introducing 3 parameters based on established growth laws for E. coli, CAFBA accurately reproduces empirical results on the growth-rate dependent rate of carbon overflow and growth yield, and generates testable predictions about cellular energetic strategies and protein expression levels. CAFBA therefore provides a simple, quantitative approach to balancing the trade-off between growth and its associated biosynthetic costs at genome-scale, without the burden of tuning many inaccessible parameters.

Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data‐independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichia coli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non‐metabolic stresses, and non‐planktonic states. The resulting high‐quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low‐abundant proteins under various metabolic limitations, the non‐specificity of catabolic enzymes upregulated under carbon limitation, the lack of large‐scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain‐dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi‐omics studies of gene regulation and metabolic control in E . coli .

Protein concentrations are set by a complex interplay between gene-specific regulatory processes and systemic factors, including cell volume and shared gene expression machineries. Elucidating this interplay is crucial for discerning and designing gene regulatory systems. We quantitatively characterized gene-specific and systemic factors that affect transcription and translation genome-wide for Escherichia coli across many conditions. The results revealed two design principles that make regulation of gene expression insulated from concentrations of shared machineries: RNA polymerase activity is fine-tuned to match translational output, and translational characteristics are similar across most messenger RNAs (mRNAs). Consequently, in bacteria, protein concentration is set primarily at the promoter level. A simple mathematical formula relates promoter activities and protein concentrations across growth conditions, enabling quantitative inference of gene regulation from omics data.

The overexpression of proteins is a major burden for fast-growing bacteria. Paradoxically, recent characterization of the proteome ofEscherichia colifound many proteins expressed in excess of what appears to be optimal for exponential growth. Here, we quantitatively investigate the possibility that this overexpression constitutes a strategic reserve kept by starving cells to quickly meet demand upon sudden improvement in growth conditions. For cells exposed to repeated famine-and-feast cycles, we derive a simple relation between the duration of feast and the allocation of the ribosomal protein reserve to maximize the overall gain in biomass during the feast.

Significance: Cell signaling involves interpreting information from the environment and implementing the appropriate regulatory control. The ppGpp signaling system is known to exert a fundamental control in bacteria, coordinating ribosome synthesis with the rate of cellular growth, but how this system can sense the cellular growth rate in different environments is unclear. Here we establish a striking linear relation between ppGpp level and the reciprocal of the translation elongation rate (ER) and show how bacteria can process complex nutritional information by monitoring this rate and using it to control ribosome synthesis. Importantly, the sensing of ER through ppGpp quantitatively and mechanistically explains bacterial growth laws which robustly relate the ribosome content and translation ERs to the growth rate.