Transcriptomic Insights into Casein-Driven Adaptive Evolution of Burkholderia thailandensis: Implications for Biofilm Formation and Antibiotic Susceptibility.
How nutrient stress shapes bacterial evolution and the associated fitness trade-offs remains a central question in microbiology. Using Burkholderia thailandensis as a surrogate for the pathogen B. pseudomallei, we performed adaptive laboratory evolution with casein as the sole carbon source. Over 30 days of serial passaging, the population shifted towards protease deficiency, with mutants constituting 54.94% by the endpoint. These evolved strains exhibited pleiotropic virulence attenuation-including reduced rhamnolipid production, motility, auto-aggregation, and biofilm formation-alongside increased susceptibility to imipenem and chloramphenicol. RNA-seq analysis of evolved strain E3101 revealed 2,836 differentially expressed genes, with significant downregulation of quorum sensing (AHL synthesis), rhamnosyltransferases, flagellar assembly, and biofilm regulatory pathways. Our findings demonstrate that casein-driven evolution selects for social 'cheaters' that conserve energy by downregulating costly virulence determinants, revealing a fundamental virulence-fitness trade-off. The coordinated transcriptional repression of biofilm and antibiotic resistance pathways provides a mechanistic framework for understanding bacterial adaptation strategies and potential therapeutic vulnerabilities in Burkholderia.