Escherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after 3 weeks of maintaining an E. coli K-12 population in stationary phase. This variant displayed a small colony phenotype and slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC(A494V) mutation confers the slow growth and small colony phenotype on this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary-phase sigma factor σ^S The RpoC(A494V) mutation upregulates the σ^S regulon. As shown over 20 years ago, early in prolonged stationary phase, σ^S attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σ^S activity, either by mutating the gene for σ^S directly or via mutations such as RpoC(A494V). The balance between the activities of the housekeeping major sigma factor and σ^S sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase.IMPORTANCE An important general mechanism of a bacterium's adaptation to its environment involves adjusting the balance between growing fast and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favor of the general stress response about 2 weeks later. This can be established by direct mutations in the master regulator of the general stress response or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance although the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.