Population reweighting and reporter distortion obscure ribosome regulation during translation inhibition

Abstract Bacteria dynamically adjust their ribosome allocation as growth conditions change, but how this regulation unfolds within individual cells remains poorly understood. Established relationships between ribosome allocation and growth are largely derived from population measurements under steady-state conditions, which may not capture the trajectories followed by individual cells during perturbation. Differential growth can reweight heterogeneous cellular states over time, causing population-level measurements to diverge from the regulatory trajectories followed by individual cells. Here, we use lineage-resolved single-cell microscopy to follow growth and ribosomal reporter dynamics in Escherichia coli during translation inhibition without growth-dependent reweighting of the sampled lineages. Unexpectedly, fluorescent protein fusions to the ribosomal proteins S2 and L9 decrease in fluorescence following chloramphenicol treatment, whereas transcriptional reporters driven by the ribosomal promoters initially increase. The decrease in ribosomal fusion fluorescence persists across multiple fluorescent proteins and is reproduced during tetracycline treatment, indicating that it is not specific to a particular fluorophore, ribosomal protein or translation inhibitor. Although some fusion reporters retain a relationship with cellular growth within physiological states, this relationship is itself reporter-dependent. These results reveal two independent requirements for quantitative measurements of dynamic cellular responses: longitudinal sampling is needed to distinguish within-cell regulation from growth-dependent population reweighting, and reporter behaviour must be validated when the perturbation alters the processes generating the measured signal.