Regulatory Evolution of TEM β-lactamases through Large-Scale Genomic Analysis Reveals Promoter Diversity and its Impact on β-lactam Resistance in Escherichia coli

Background: Antimicrobial resistance is a major global health threat, with β-lactamases representing the most prevalent mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria. Among these, TEM β-lactamases ( bla _TEM ) are the earliest identified and most widely disseminated resistance determinants in Escherichia coli . Given the established influence of canonical promoter variation on bla _TEM expression and β-lactam resistance, evolutionary diversification of the bla _TEM upstream regulatory region can affect the resistance phenotype. However, the extent to which upstream variation is associated with resistance beyond changes in the coding sequence remains unclear. Results: We analyzed 60,087 E. coli genomes from BV-BRC to characterize bla _TEM upstream regulatory regions, focusing on -35 and -10 promoter motifs and their relationship with β-lactam resistance. P3 was the prevalent canonical promoter, while multiple non-canonical promoter types with distinct -10 and -35 motifs were identified. A causal-inference framework estimated promoter-associated differences in resistance probability while holding the underlying bla _TEM variant profile constant. Average treatment effect estimates showed that several P3 transitions were associated with increased predicted resistance probabilities across multiple β-lactam antibiotics. Novel motifs from single- bla _TEM isolates were further prioritized by occurrence frequency and evaluated structurally with σ⁷⁰ DNA-recognition domains using molecular dynamics simulations. Notably, the -35 TGCAAC motif maintained consistent σ⁷⁰ interactions, supporting its structural compatibility as a putative promoter motif potentially relevant to bla _TEM regulation and resistance-associated phenotypes. Conclusion: Overall, this study integrates large-scale genomics, causal inference, and structural analysis to investigate regulatory variation alongside coding-sequence changes, providing insight into the evolving mechanisms of antimicrobial resistance.