Hypoxia drives asymmetric envelope remodelling to promote membrane adaptation and antibiotic tolerance in mycobacteria

Abstract Mycobacterium tuberculosis persists within the host by adapting to stressful microenvironments, with hypoxia representing a key clinically relevant challenge. However, how hypoxia alters mycobacterial membrane organization and function remains poorly understood. Using Mycobacterium smegmatis as a model, we combined membrane biophysics, layer-resolved lipidomics, and membrane-drug interaction analyses to characterize envelope adaptation under oxygen limitation. Hypoxia induced asymmetric remodelling of the cell envelope, with the inner membrane becoming more ordered and less hydrated, while the outer membrane exhibited increased fluidity and hydration. Lipidomic analyses revealed corresponding membrane-specific changes, including enrichment of saturated, tightly packed lipids in the inner membrane and unsaturated, inverted-conical lipids in the outer membrane. Functionally, hypoxic cells showed reduced envelope permeability and decreased susceptibility to rifampicin, while membrane partitioning studies suggested reduced rifampicin association with hypoxia-adapted inner membranes. We identify asymmetric envelope remodeling as a previously unrecognized principle of bacterial adaptation to environmental stress. Rather than responding as a uniform structure, the mycobacterial envelope undergoes coordinated but divergent remodeling in its constituent membrane layers during hypoxia. This spatially resolved response couples inner membrane rigidification with outer membrane fluidization, generating a functionally specialized envelope architecture that simultaneously preserves membrane integrity, modulates permeability, and reduces antibiotic association. These findings suggest that membrane asymmetry is not merely a structural feature of complex bacterial envelopes but an actively regulated adaptive strategy that links environmental sensing to stress resilience and antimicrobial tolerance Significance Most studies view bacterial membranes as responding uniformly to environmental stress. By resolving the inner and outer membranes separately, we show that hypoxia drives opposing remodelling programs within the mycobacterial envelope. This asymmetric adaptation alters membrane organization, permeability, and rifampicin interactions, revealing a previously unrecognized mechanism of physiological adaptation and antibiotic tolerance. The findings suggest that membrane asymmetry itself constitutes an important regulatory feature of pathogen survival under stress and may provide new opportunities for therapeutic intervention against persistent mycobacterial infections.