Experimental evolution reveals biphasic adaptation of Bacillus subtilis to long-chain fatty acid stress

Gram-positive bacteria like Bacillus subtilis are typically sensitive to long-chain fatty acids (LCFAs), which serve as both nutrients and antimicrobials, posing an ecological paradox regarding their survival in lipid-rich environments. We experimentally evolved B. subtilis for ~ 1,500 generations under progressively increasing oleic acid stress, monitoring growth, morphology, and genomic changes. Initial exposure induced reversible cell chaining, suggesting a plastic morphological response that may help buffer oleate toxicity. Over time, this trait became genetically fixed, with convergent mutations identified in genes related to cell division, peptidoglycan synthesis, and regulation. These findings reveal a biphasic adaptation where phenotypic plasticity buffers early stress, followed by genetic accommodation that facilitates survival and provides insights into how Gram-positive bacteria adapt to lipid stress, potentially informing antimicrobial strategies.