The evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells., Microbial evolution often involves transient phenotypes and sequential development of multiple mutations of unclear relevance. Here, the authors show that the evolution of non-growing E. coli cells can be driven by alterations in pyrimidine nucleoside levels associated with colony ageing and/or due to mutations in metabolic or regulatory genes.