Bacterial growth under confinement requires transcriptional adaptation to resist turgor pressure build-up.

Bacterial proliferation in confined spaces occurs in biofilms, intracellular compartments, or infection sites, generating mechanical constraints. We investigated how growth-induced mechanical pressure affects bacterial physiology using a microfluidic device ensuring nutrient access. We found that proliferating Escherichia coli cells generate forces in the hundreds of kPa range decoupling growth and division, producing shorter bacteria. Increased cytoplasmic protein concentrations and crowding induce division arrest and decreased protein synthesis. In contrast, growth-induced pressure keeps increasing during confinement. Theoretical modeling predicts this novel regime of steady pressure increase, termed overpressurization, driven by persistent metabolite synthesis. Under confinement, the Rcs pathway is activated, and rcs mutants display abnormal shapes only in the overpressurized state. Therefore, transcriptional adaptation is required to resist pressure build-up. A uropathogenic strain displayed the same confined growth phenotypes in vitro, highlighting the potential relevance of these pressurized regimes during infection.