Genomic diversification, adaptive convergence, and regulatory rewiring in aging Escherichia coli colonies

Bacterial colonies are dynamic evolutionary microenvironments where spatial heterogeneity and nutrient gradients generate diverse ecological niches. Understanding how such structured environments drive genetic and functional diversification, including the emergence of clinically relevant traits such as antibiotic resistance, remains a major challenge. To dissect adaptive strategies in structured populations, we performed whole-genome sequencing on 24 Escherichia coli isolates recovered from a three-week-old colony. Transcriptomic profiling was conducted on two representative mutants, and targeted competition assays were used to assess fitness relative to the parental strain. Genome sequencing uncovered extensive evolutionary diversification, with 34 distinct mutations, mostly insertion-sequence events, affecting transcriptional, stress-response, metabolic, and envelope regulators. Strikingly, half of all isolates carried mutations in the yobF-cspC operon, identifying it as a major adaptive hotspot in structured populations. Transcriptomic analyses revealed a broad regulatory shift in yobF-cspC mutants, with substantial activation of central metabolism and biosynthesis coupled to repression of acid resistance and stress pathways. Functionally, deletion of cspC alone conferred a robust σS-independent fitness advantage and fully restored competitiveness in an rpoS-inactivated background in aging colonies. Beyond this dominant adaptive trajectory, genome analysis revealed isolates carrying mutations that confer gain-of-function phenotypes, including β-lactam and rifamycin resistance, despite the absence of antibiotic pressure, highlighting aging colonies as potential reservoirs of clinically relevant diversity. Our results suggest that aging bacterial colonies can serve as incubators of evolutionary innovation, simultaneously generating diverse functional variants while selecting for metabolic specialization. Together, these findings show how spatial structure and nutrient recycling shape bacterial adaptation and diversification, providing a powerful and tractable model to investigate evolutionary processes in natural and pathogenic communities.