New insights into the structure-based mechanism of Bacillus subtilis spore resistance to high hydrostatic pressure.

The exceptional pressure resistance of bacterial spores limits the application of high hydrostatic pressure (HHP) in food sterilization, yet the underlying mechanisms remain poorly understood. Here, we examined how structural modifications (coat, cortex, inner membrane [IM], α/β-type small acid-soluble proteins [α/β-type SASPs]) in Bacillus subtilis spores affect HHP-induced germination, superdormant (SD) subpopulation, and inactivation, which together define HHP resistance. Key findings reveal that (i) coat defects likely alleviate cortex compression on IM, thereby influencing germination-associated proteins. This alteration reduces germination efficiency at 200 MPa but enhances it at 500 MPa, leading to an expanded SD subpopulation and increased resistance at 200 MPa, while a diminished SD ratio and reduced resistance at 500 MPa; (ii) increased cortex cross-linking combined with core expansion potentially modifies IM properties, strongly affecting germination-related proteins. This change markedly promotes germination under both 200 and 500 MPa, resulting in decreased SD ratios and thus lower spore resistance under both conditions; (iii) reduced cardiolipin content in IM likely alters its biophysical properties, specifically increasing germination efficiency at 500 MPa. This contributes to a lower SD ratio and reduced resistance at 500 MPa; (iv) the absence of α/β-type SASPs has minimal effect on resistance at 500 MPa but reduces resistance at 200 MPa by decreasing the SD ratio, potentially due to spontaneous germination after treatment. Collectively, HHP resistance is modulated by multiple structural components through distinct mechanisms. Structural modifications that alter IM biophysical properties or the function of germination-related proteins appear to be critical regulators of pressure-induced germination.IMPORTANCEOur findings elucidate the structural mechanism underlying the exceptional pressure resistance of bacterial spores, which represent a major challenge for high hydrostatic pressure sterilization. This study establishes a structure-based framework that not only clarifies the biophysical principles of spore resilience but also provides a foundation for developing novel strategies to achieve mild spore inactivation in the food industry. Beyond these practical implications, our work offers fundamental theoretical insights into microbial pressure resistance mechanisms.