Regulatory mutation accumulation drives multi-trait phenotypic expansion in silver-adapted Escherichia coli

Predicting when adaptive mutations produce narrow, condition-specific effects versus coordinated multi-trait phenotypic expansion remains a central challenge in evolutionary biology. Two-component response systems (TCRSs) play a key role in bacterial environmental sensing and transcriptional regulation, yet how regulatory variation shapes broader adaptive outcomes remains poorly understood. Here, we reanalyze and integrate our previously published experimental evolution, whole-genome sequencing, and transcriptomic data with new high-throughput phenotypic profiling, and competitive fitness assays to examine how regulatory context influences multi-trait responses during bacterial adaptation. Using silver-adapted Escherichia coli populations, we show that independently evolved lineages carrying cusS (a TCRS with CusR that regulates copper/silver efflux) mutations, along with additional regulatory mutations, exhibit broader phenotypic expansion across metabolic and stress-response conditions, whereas engineered single cusS mutations produce constrained phenotypic responses. Comparative transcriptomic analyses reveal that evolved populations display expanded engagement of functional pathways relative to engineered mutants, consistent with increased regulatory diversification. However, competitive fitness assays against an engineered single mutant revealed that this phenotypic expansion was not uniformly adaptive: four of six evolved lineages fell below parity against the engineered mutant, while two lineages, despite comparable phenotypic breadth, did not. Together, these results indicate that multi-trait phenotypic expansion during adaptation is associated with regulatory mutation accumulation and transcriptional remodeling rather than with isolated adaptive mutations, but that this expansion does not reliably predict competitive fitness outcome. More broadly, our findings highlight the importance of regulatory architecture in shaping evolutionary trajectories and underscore the need for direct fitness measurements when interpreting coordinated phenotypic responses in microbial evolution.

Bacteria constantly encounter stresses, such as antibiotics and toxic metals, and must adapt to survive. While individual genetic changes can help bacteria tolerate stress, it remains unclear why some adaptations affect only a single trait while others reshape many aspects of physiology, and whether these broader changes make bacteria more competitive or simply reflect disorganized regulation. Here, we combined evolutionary experiments, genomic and transcriptomic data, phenotypic profiling, and head-to-head competition assays to show that broad changes in bacterial behavior are associated with accumulation of regulatory mutations rather than single adaptive changes, but do not always confer a real competitive advantage. Our results show that regulatory network structure shapes how widely adaptation impacts metabolism, stress tolerance, and cellular function, while also showing that phenotypic breadth alone does not reliably predict fitness. These findings advance our understanding of bacterial evolution and may inform efforts to manage microbial responses to clinical stressors.