SRA

During steady-state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here, we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in the bacterium Bacillus subtilis. While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom-up models of expression-fitness landscapes will require near-exhaustive characterization Overall design: 26 competition experiment amplicon libraries from strain pools, 12 matched Rend-seq and ribosome profiling datasets, one individual Rend-seq dataset, 56 RNA-seq datasets. All data from Bacillus subtilis samples.